Book OilCrops

Oil Crops
HANDBOOK OF PLANT BREEDING
Editors-in-Chief:
JAIME PROHENS, Universidad Politecnica de Valencia, Valencia, Spain
FERNANDO NUEZ, Universidad Politecnica de Valencia, Valencia, Spain
MARCELO J. CARENA, North Dakota State University, Fargo, ND, USA
Volume 1
Vegetables I: Asteraceae, Brassicaceae, Chenopodicaceae, and Cucurbitaceae
Edited by Jaime Prohens and Fernando Nuez
Volume 2
Vegetables II: Fabaceae, Liliaceae, Solanaceae and Umbelliferae
Edited by Jaime Prohens and Fernando Nuez
Volume 3
Cereals
Edited by Marcelo J. Carena
Volume 4
Oil Crops
Edited by Johann Vollmann and Istvan Rajcan
Johann Vollmann l
Istvan Rajcan
Editors
Oil Crops
13
Editors
Johann Vollmann Istvan Rajcan
Institute of Agronomy & Plant Breeding Department of Plant Agriculture
BOKU-University of Natural University of Guelph
Resources & Applied Life Sciences 50 Stone Road W.
Gregor Mendel Str. 33 ON N1G 2W1
1180 Vienna Crop Science Bldg.
Austria Canada
johann.vollmann@boku.ac.at irajcan@uoguelph.ca
ISBN 978-0-387-77593-7 e-ISBN 978-0-387-77594-4

DOI 10.1007/978-0-387-77594-4
Springer Dordrecht Heidelberg London New York
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Foreword
When one is privileged to participate long enough in a professional capacity,

certain trends may be observed in the dynamics of how challenges are met or
how problems are solved. Agricultural research is no exception in view of how
the plant sciences have moved forward in the past 30 years. For example, the
once grand but now nearly forgotten art of whole plant physiology has given
way almost completely to the more sophisticated realm of molecular biology.
What once was the American Society of Plant Physiologists’ is now the American
Society of Plant Molecular Biology; a democratic decision to indemnify efforts
to go beyond the limits of the classical science and actually begin to understand
the underlying biological basis for genetic regulation of metabolic mechanisms
in plants. Yet, as new technologies open windows of light on the inner workings
of biological processes, one might reminisce with faint nostalgia on days long
past when the artisans of plant physiology, biochemistry, analytical chemistry
and other scientific disciplines ebbed and waned in prominence.
No intentional reference is made here regarding Darwinism; the plant
sciences always have been extremely competitive. Technology is pivotal.
Those who develop and/or implement innovative concepts typically are
regarded as leaders in their respective fields. Each positive incremental step
helps bring recognition and the impetus to push a scientific discipline forward
with timely approaches to address relevant opportunities.
So, it might be interesting to know how those skilled in the art of statistical
analysis and the field of classical plant quantitative genetics are coping with the
intensifying research emphasis on biotechnology, genomics, proteomics, and
the like. After all, high-throughput whole genome sequence analyses and
advanced bioinformatic resources for gene discovery will soon render the
characterization of haplotypes, in entire germplasm collections and among
progeny of segregating breeding populations, a routine event. Will the day
come when breeders are told which parents to mate for a particular objective?
No doubt an interesting dialog will ensue, but by-in-large taking the mystery
out of plant science should be viewed as a good thing for all the constituent
professions.
Is a physician’s ability impaired by the advent of new diagnostic technologies
and a more effective range of pharmaceuticals? Even a NASCAR driver
v
vi Foreword
benefits from all of the computerized signals that monitor every aspect of a
race cars performance. So, it is the same for breeding and quantitative
genetics. Knowledge and skill are still needed to associate phenotypic traits
with a haplotype. Ability is still required to reduce all of these ancillary tools
to successful practice. Thus, the renaissance that is underway will position
plant quantitative genetics to emerge with increased capacity to provide
solutions to major problems and address the needs of world agriculture in a
timely manner.
What are those needs with regard to oilseeds? Based on world production,
USDA Foreign Agricultural Service reports show that soybean (56.0%),
rapeseed (13.4%), cottonseed (10.1%), peanut (8.1%), sunflower (8.0%), and
palm plus palm kernel (2.8%) are the major oilseed crops. These commodities
represent essentially the entire commercial source of vegetable protein and oil.
Annual world consumption of vegetable oil has averaged about 90.0% of total
vegetable oil supply since 1997, leaving on average enough end-of-year stocks
for about a 30-day buffer; whereas annual world use of oilseed meal has
averaged about 95.7% of total supply, leaving on average a carryover
equivalent to about an 11-day cushion of meal. These trends suggest that
consumer demand for these products is limited only by availability, and that
any natural disaster that may limit oilseed production could severely
compromise the global food chain.
Although crushing capacity has expanded significantly in the US and abroad,
the proportion crushed has averaged about 81% of total world oilseed
production for decades. Considering the need to service export markets, a
significant escalation of oilseed crush levels to increase the supply of meal and
oil is unlikely. Hence, the greatest need that oilseed breeders face is simply to
ensure a sufficient oilseed supply to meet the elastic demand for protein and oil;
which on its own merit is a major contribution to alleviate world hunger.
However in recent years, a number of constraints have emerged that could
mitigate efforts to increase
global oilseed production 100 25
for food use. The most World Vegetable Oil Consumption

World Food Use Vegetable Oils
World Industrial Use Vegetable
prominent factor is 95
Oils (% Total Consumption)
19.8
(% Total Consumption)
renewed interest in Food Use 20

vegetable oil as a source 90 89.3
90.5 17.7
of biodiesel fuel. This 89.8 87.5
concern recognizes that 85 15
annual global vegetable 13.8
Industrial Use 82.3
oil resources could barely 80
80.2
11.5
make a dent in the demand 10.2
Growth in Demand 10
for energy. However, as 9.5
5.26 MMT/yr
75 2
shown in the adjacent R = 0.992
figure, the market forces USDA FAS, Jan 2009

70 5
that direct food and
1995 2000 2005 2010
industrial demand for Year
Foreword vii
vegetable oils appear to have established a temporary equilibrium at about 80%

(food):20% (industrial). Perhaps this will hold long enough for appropriate
adjustments in markets for oilseed products. In addition, breeding efforts to
develop varieties for commercial production of industrial oilseeds like
lesquerella, cuphea and various non-food biotech innovations should help
stabilize this situation.
Achieving greater genetic gain for oilseed productivity may be a lesser
priority to some in the oilseed industry who subscribe to the paradigm that
farmers will expand harvested area to increase the production of oilseeds.
However, in view of escalating costs of oilseed production and competition
for land from non-oilseed crops, the flexibility of countries to devote more
agricultural resources to oilseeds remains to be seen. At this time, the rate of
increase in harvested area since 1997 may be the best estimate of how much
more harvested area might be available in future years. Regression analysis of
these data in the figure below estimates the rate of increase at +3.45 Mha per
year (R2, 0.88). Assuming continuation of a linear trend, there might be a total
of 258 Mha in global oilseed production in the year 2020, an increase of about
41 Mha over the level in 2008. One must wonder if this would be enough to
make a significant difference.
425 2050
Questions about future
levels of harvested area
400
1950 place more pressure on
World Oilseed Yield (kg/ha)
World Oilseed Production
375 Production
(MMT) or Harvested Mha
1850
the remaining variable in
350
the yield equation for
325 Yielding
Ability
1750 increased production.
300 Regression analysis of
1650
275 these data in the adjacent
250 1550 figure estimates the rate of
225 increase in world oilseed
Land 1450
200
production at +12.5
175
1350 MMT per year (R2, 0.96).
Assuming continuation of
150 1250
1995 1998 2001 2004 2007 2010
a linear trend, there might
Year be a total of 704 MMT in
global oilseed production
in the year 2020, an increase of about 196 MMT over the level in 2008. Again,
using simple arithmetic and assuming 258 Mha would be available to harvest, the
world average oilseed yield in 2020 could be about 2.7 MT per ha (or 3.2 MT per ha
if no additional land became available). Reaching that plateau would require a
40% increase in average total oilseed yield (70% without the projected increase in
land) given an average global oilseed yield of 1.9 MT per ha in 2008.
In the past decade, average global oilseed yield has increased only 20%.
Therefore, it appears that a great deal is riding on the development and
application of oilseed biotechnology and genomics in the next decade. These
technologies should enable quantum leaps in genetic progress. However, it all
viii Foreword
depends upon a renaissance in quantitative genetics and the application of those

technologies now and by the next generation of public and private oilseed
breeders. Perhaps, it would be wise to redouble the effort to train and deploy
that future workforce now.
Raleigh, North Carolina Richard F. Wilson

Preface
Vegetable oils have gained in importance during the past few decades resulting
in the doubling of the world oil crop production in the last 25 years. Oil crops
have been increasingly used as raw materials for food, livestock feed and non-
food industrial applications. Plant breeding has played an essential role in
supporting these developments: Breeding for higher yield and oil content
allowed for an increase in oil production per unit area, whereas breeding for
better oil quality has improved both the human health value of vegetable oils as
well as the suitability of particular oils in specific industrial applications.
Moreover, newly developed unique oil qualities are opening new
opportunities in agricultural production and processing.
Cereals, legumes or forages each represent relatively homogeneous groups of
crops belonging to one or a few plant families with similar botanical
characteristics in which comparable breeding procedures could be used. In
contrast, oil crop species have been developed in various botanical families
from both the monocots and dicots. Thus, oil crops are a highly diverse set of
species from short season annuals to perennials with a life span of over 2000
years. Consequently, breeding methods used for oil crop improvement include
clonal breeding, pure line breeding, improvement of open-pollinated
populations as well as hybrid breeding. In particular, the breeding procedures
and techniques include almost every activity from simple mass selection and
hybridization to specialized biotechnologies such as in vitro propagation or
genetic engineering. Despite the differences at the species and breeding levels,
some major breeding goals are remarkably similar, which justifies treating them
in one volume such as: high oil content, altering fatty acid composition to suit
the needs for either human consumption or non-food utilization, and a high
quality of by-products. In addition, issues such as the biosynthetic pathways of
particular fatty acids and their manipulation, QTL analysis for quality
characters, genetic diversity, or oil and fatty acid analytics during selection
are of common interest to all oil crop breeders. Therefore, this volume was
prepared as a state-of-the-art compilation and a major reference text on oil crop
breeding, which has been lacking for several decades. While the information
accumulated in this volume is of primary interest to plant breeders, valuable
insights are also offered to agronomists, molecular biologists, physiologists,
ix
x Preface
plant pathologists, food scientists and university scholars from the comparative
treatment of various oil crop species.
Apart from an introductory chapter on oil crop breeding and a chapter
highlighting genetic modification of vegetable oils, this volume presents 17
chapters devoted to breeding of particular oil crop species. Oil crops with
world-wide distribution such as soybean, sunflower, oilseed rape and related
brassicas are presented side-by-side with tropical and subtropical species such
as cotton seed, peanut or castor, the perennials oil palm, coconut and olive,
minor oil crops of regional importance such as safflower, poppy, oil pumpkin or
maize, and new oil crops such as lesquerella and cuphea. Origin and
domestication, varietal groups, genetic resources, major achievements and
current breeding goals, breeding methods, techniques and biotechnologies,
and seed production are addressed depending on their relevance in a
particular crop.
Each crop chapter has been written by outstanding experts in their respective
fields. Whenever possible authors from different institutions or countries
worked together on particular chapters, which contributed to broadened and
well-balanced views on particular species or topics.
The editors acknowledge the excellent contributions of all chapter authors
who devoted much time and effort in delivering their part to this high quality
volume. The editors extend heartfelt thanks to the staff at Springer, particularly
to Hannah Schorr and Jinnie Kim, for their highly professional support during
all stages of the publishing process. Moreover, the editors would like to thank
Editors-in-chief of the Springer series Handbook of Plant Breeding, Professors
Jaime Prohens, Fernando Nuez and Marcelo Carena, both for considering a
volume exclusively devoted to oil crops and for their helpful input throughout
the preparation of this volume.
Vienna, Austria Johann Vollmann

Guelph, ON, Canada Istvan Rajcan
Contents
1 Oil Crop Breeding and Genetics . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1

Johann Vollmann and Istvan Rajcan
2 Modifying Vegetable Oils for Food and Non-food Purposes . . . . . . . 31

Edgar B. Cahoon, Thomas E. Clemente, Howard G. Damude,
and Anthony J. Kinney
3 Soybean . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 57
Elroy R. Cober, Silvia R. Cianzio, Vincent R. Pantalone,
and Istvan Rajcan
4 Oilseed Rape . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 91
Wolfgang Friedt and Rod Snowdon
5 Other Brassicas . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 127

Leonardo Velasco and José M. Fernández-Martı́nez
6 Sunflower . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 155
José M. Fernández-Martı́nez, Begoña Pérez-Vich,
and Leonardo Velasco
7 Flax . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 233
Scott D. Duguid
8 Cotton . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 257
Steve Hague, Lori Hinze, and James Frelichowski
9 Peanut . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 287
Barry L. Tillman and H. Thomas Stalker
10 Castor . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 317
Dick L. Auld, Mauricio D. Zanotto, Thomas McKeon,
and John B. Morris
xi
xii Contents
11 Oil Palm. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 333

Aik Chin Soh, Choo Kien Wong, Yuk Wah Ho,
and Chieh Wean Choong
12 Coconut . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 369
Lalith Perera, Suriya A.C.N. Perera, Champa K. Bandaranayake,
and Hugh C. Harries
13 Olive. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 397
Luciana Baldoni and Angjelina Belaj
14 Safflower . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 423
Hans-Henning Mündel and Jerald W. Bergman
15 Poppy . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 449
Jenö Bernáth and Éva Németh
16 Hull-Less Oil Seed Pumpkin . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 469

Tamás Lelley, Brent Loy, and Michael Murkovic
17 Maize for Oil . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 493

Elizabeth A. Lee
18 New Crops Breeding: Lesquerella. . . . . . . . . . . . . . . . . . . . . . . . . . . . 507

David Dierig and Dennis T. Ray
19 Cuphea . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 517
Winthrop B. Phippen
Index . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 535
Contributors
Dick L. Auld Department of Plant and Soil Science, Texas Tech University,
Food Technologies, Lubbock, TX 79409-3121, USA, dick.auld@ttu.edu
Luciana Baldoni CNR, Istituto di Genetica Vegetale, Via Madonna Alta, 130,
06128 Perugia, Italy, luciana.baldoni@igv.cnr.it
Champa K. Bandaranayake Department of Primary Industries, Plant Genetics
and Genomics, Hamilton, Victoria 3300, Australia,
champa.bandaranayake@dpi.vic.gov.au
Angjelina Belaj IFAPA, Centro ‘‘Alameda del Obispo’’, Avda Menéndez Pidal
s/n, 14083 Cordoba, Spain, angjelina.belaj.axt@juntadeandalucia.es
Jerald W. Bergman Eastern Agric. Res. Center, Montana State University,
Agric. Extension Service, Sidney, MT 59270, USA,
jbergman@sidney.ars.usda.gov
Jenö Bernáth Department of Medicinal and Aromatic Plants, Corvinus
University of Budapest, H-1118 Budapest, Hungary, jeno.bernath@uni-
corvinus.hu
Edgar B. Cahoon The Donald Danforth Plant Science Center, St. Louis, MO
63132, USA, ecahoon@danforthcenter.org
Chieh Wean Choong Applied Agricultural Resources, Sg. Buloh, Selangor;
INTI University College, Persiaran Perdana BBN, Putra Nilai, 71800 Nilai,
Negri Sembilan, Malaysia, choong_chiehwean@intimal.edu.my
Silvia R. Cianzio Department of Agronomy, Iowa State University, Ames, IA
50011-1010, USA, scianzio@iastate.edu
Tom E. Clemente Department of Horticulture and Agronomy, Center for
Plant Science Innovation, University of Nebraska, Lincoln, NE 68588-0660,
USA, tclemente1@unl.edu
Elroy R. Cober Agriculture and Agri-food Canada, Eastern Cereal and Oilseed
Crop Research Centre, Ottawa, ON, K1A 0C6, Canada,
coberer@agr.gc.ca
xiii
xiv Contributors
Howard G. Damude DuPont Experimental Station, Crop Genetics Research

and Development, Wilmington, DE 19880-0353, USA,
howard.g.damude@usa.dupont.com
David Dierig USDA-ARS-Arid Lands Agricultural Research Center, 21881
North Cardon Lane, Maricopa, AZ 85239, USA, david.dierig@ars.usda.gov
Scott D. Duguid Morden Research Station, Agriculture and Agri-Food
Canada, Morden, MB, Canada R6M1Y5, scott.duguid@agr.gc.ca
José M. Fernández-Martı́nez Institute for Sustainable Agriculture (CSIC),
Alameda del Obispo s/n, 14004 Córdoba, Spain, cs9femaj@uco.es
James Frelichowski Crop Germplasm Research Unit, USDA-ARS-SPARC,
TX 77845, USA, jfrelichowski@sparc.usda.gov
Wolfgang Friedt Department of Plant Breeding, Land Use and Nutrition, IFZ
Interdisciplinary Research Centre for BioSystems, Justus-Liebig-University of
Giessen, Heinrich-Buff-Ring 26-32, D-35392 Giessen, Germany,
wolfgang.friedt@agrar.uni-giessen.de
Steve Hague Department of Soil and Crop Sciences, Texas A&M University,
2474 TAMU, College Station, TX 77843-2474, USA, shague@ag.tamu.edu
Hugh C. Harries Coconut Industry Board of Jamaica (retired), Weymouth,
Dorset, DT3 5NP, UK, hugh.harries@gmail.com
Lori Hinze Crop Germplasm Research Unit, USDA-ARS-SPARC, TX 77845,
USA, lori.hinze@ars.usda.gov
Yuk Wah Ho Applied Agricultural Resources, 47000 Sg. Buloh, Selangor,
Malaysia, aarsb@aarsb.com.my
Anthony J. Kinney DuPont Experimental Station, Crop Genetics Research
and Development, Wilmington, DE 19880-0353, USA,
anthony.kinney@usa.dupont.com
Elizabeth A. Lee Department of Plant Agriculture, University of Guelph, Crop
Science Bldg., Guelph, ON N1G 2W1, Canada, lizlee@uoguelph.ca
Tamás Lelley Department of Agrobiotechnology Tulln, University of Natural
Resources and Applied Life Sciences Vienna (BOKU), Konrad Lorenz Strasse
20, A-3430 Tulln, Austria, tamas.lelley@boku.ac.at
Brent Loy Department of Biological Sciences, University of New Hampshire,
Durham, NH 03824, USA, jbloy@cisunix.unh.edu
Tom A. McKeon USDA-ARS, PWA, WRRC-CIU, Albany, CA, 94710, USA,
tmckeon@pw.usda.gov
John B. Morris USDA-ARS, Griffin, GA 30223, USA,
brad.morris@ars.usda.gov
Contributors xv
Hans-Henning Mündel West View Mall, Lethbridge, AB T1K 6X4, Canada,

hhmuend@shockware.com
Michael Murkovic Institute for Food Chemistry and Technology, Graz
University of Technology, Petersgasse 12, A-8010 Graz, Austria,
michael.murkovic@tugraz.at
Éva Németh Department of Medicinal and Aromatic Plants, Corvinus
University of Budapest, H-1118 Budapest,
Hungary, eva.nemeth@uni-corvinus.hu
Vincent R. Pantalone Department of Plant Sciences, University of Tennessee,
Knoxville, TN 37996-4561, USA, vpantalo@utk.edu
Lalith Perera Genetics and Plant Breeding Division, Coconut Research
Institute of Sri Lanka, Lunuwila, Sri Lanka, kanthaperera@yahoo.com;
chandrikaperera2003@yahoo.com
Suriya A.C.N. Perera Genetics and Plant Breeding Division, Coconut
Research Institute of Sri Lanka, Lunuwila, Sri Lanka,
chandrikaperera2003@yahoo.com; kanthaperera@yahoo.com
Begoña Pérez-Vich Institute for Sustainable Agriculture (CSIC), Alameda del
Obispo s/n, 14004 Córdoba, Spain, bperez@cica.es
Winthrop B. Phippen Department of Agriculture, Western Illinois University,
1 University Circle, Macomb, IL 61455, USA, wb-phippen@wiu.edu
Istvan Rajcan Department of Plant Agriculture, University of Guelph,
Guelph, ON N1G 2W1, Canada,
irajcan@uoguelph.ca
Dennis T. Ray Department of Plant Sciences, Division of Horticultural and
Crop Sciences, The University of Arizona, Tucson, AZ 85721, USA,
dtray@email.arizona.edu
Rod Snowdon Department of Plant Breeding, IFZ Interdisciplinary Research
Centre for BioSystems, Justus-Liebig-University of Giessen, Land Use and
Nutrition, Heinrich-Buff-Ring 26-32, D-35392 Giessen, Germany,
rod.snowdon@agrar.uni-giessen.de
Aik Chin Soh Applied Agricultural Resources, UNMC Biotechnology
Research Centre, Jalan Broga, 43500 Semenyih, Selangor, Malaysia,
sohac28@gmail.com
H. Thomas Stalker Department of Crop Science, North Carolina State
University, Raleigh, NC 27695-7629, USA, tom_stalker@ncsu.edu
Barry L. Tillman Agronomy Department, North Florida REC, University of
Florida, Marianna, FL 32446-7906, USA, btillman@ufl.edu
xvi Contributors
Leonardo Velasco Institute for Sustainable Agriculture (CSIC), Alameda del

Obispo s/n, 14004 Córdoba, Spain, ia2veval@uco.es
Johann Vollmann Institute of Agronomy and Plant Breeding, University of
Natural Resources and Applied Life Sciences Vienna (BOKU), 1180 Vienna,
Austria, johann.vollmann@boku.ac.at
Choo Kien Wong Applied Agricultural Resources, Sdn. Bhd., 47000 Sg. Buloh,
Selangor, Malaysia, aarsb@aarsb.com.my
Mauricio D. Zanotto Department of Crop Science, Sao Paulo State University,
Rua José Barbosa de Barros, 1780, Fazenda Lageado, Cx. P. 237, 18603-970
Botucatu, SP, Brazil, zanotto@fca.unesp.br
Chapter 1
Oil Crop Breeding and Genetics
Johann Vollmann and Istvan Rajcan
1.1 Introduction
Oil crops have considerably gained in importance to world agriculture and

associated industries over the past 25 years. The total area of land devoted to oil
crop cultivation has seen an increase from 160 million hectares in 1980 to 247
million hectares in 2005 (Fig. 1.1), whereas the world-wide acreage of cereals
has dropped from 717 to about 670 million hectares over the same period of
time. Annual world oil crop production has risen from 278 million metric
tonnes in 1980 to about 711 million metric tonnes in year 2005 (Fig. 1.1). This
remarkable expansion of production is due to the process of concentration
on major oil crop species and at the same time to yield increases per unit area
through refined agronomic practice and plant breeding. As illustrated in
Table 1.1, soybean, rapeseed, sunflower and oil palm are the major crops
contributing to the increase of the overall oil crop cultivation area, whereas
the acreages of cotton seed, linseed, safflower and castor had significant
decreases. Increases in yield per unit area from the 1979–1981 period to the
2002–2004 period (Table 1.1) were 82.2 and 69.0% for oil palm and rapeseed,
respectively, and were also high for linseed and castor. A more moderate yield
increase from 1701 to 2284 kg/ha (i.e. 34.3%) was noticeable for soybean,
whereas progress was very slow in sunflower and safflower, and even negative
in poppy. Taking the 2005/2006 marketing year and the medium-term pro-
spects assessment for agricultural commodities of FAO and OECD as a basis,
world oil crop production and vegetable oil output were estimated to rise by
another 25–30% by the year 2015 (Thoenes 2006). Projections for the period
2006–2015 show that production increases will slow down in Europe and
North America, while they will notably grow in Brazil, Argentina, Malaysia
and Indonesia. Both the oil crop production increases since 1980 and the
projected growth until 2015 correspond with a steadily growing demand for
J. Vollmann (*)
University of Natural Resources and Applied Life Sciences Vienna (BOKU),
Institute of Agronomy and Plant Breeding, Vienna, Austria
e-mail: johann.vollmann@boku.ac.at
J. Vollmann, I. Rajcan (eds.), Oil Crops, Handbook of Plant Breeding 4, 1

DOI 10.1007/978-0-387-77594-4_1, Ó Springer ScienceþBusiness Media, LLC 2009
2 J. Vollmann and I. Rajcan
Fig. 1.1 Global oil crop production and acreage from 1980 to 2005 (FAOSTAT 2007)
Table 1.1 Changes in world acreage and average annual seed or fruit yield of major oil crops
over a 25 years period of time
Area in hectares Change Average yield in kg/ha Change
Species 1980 2005 (in %) 1979–1981 2002–2004 (in %)
Castor 1, 540, 418 1, 408, 773 –8.5 572 934 63.1
Coconut 8, 768, 644 10, 685, 108 21.9 3, 717 4, 994 34.4
Cotton seed 34, 523, 000 30, 000, 000 –13.1 1, 246 1, 821 46.1
Groundnut 18, 364, 563 23, 427, 479 27.6 988 1, 452 47.0
Linseed 5, 371, 117 3, 182, 058 40.8 456 767 68.2
Oil palm 4, 277, 328 12, 395, 528 189.8 7, 052 12, 847 82.2
Olive 5, 130, 401 7, 550, 561 47.2 1, 853 2, 042 10.2
Poppy 58, 573 109, 164 86.4 579 504 12.9
Rapeseed 10, 992, 015 27, 448, 263 149.7 970 1, 639 69.0
Safflower 1, 322, 348 916, 443 30.7 694 741 6.7
Sesame 6, 265, 283 7, 279, 469 16.2 303 435 43.7
Soybean 50, 649, 297 92, 369, 299 82.4 1, 701 2, 284 34.3
Sunflower 12, 425, 559 22, 823, 330 83.7 1, 170 1, 225 4.7
Total 160, 618, 770 241, 961, 583 50.6
Source: FAOSTAT 2007.
and consumption of vegetable oils and fats. From 1980 to 2003, the avail-
ability of vegetable oils for food was rising from 19.9 to 26.4 kg per caput per
year for North America and from 11.6 to 19.6 kg for Western Europe, whereas
it developed from 4.8 to 10.3 kg for Asia and from 7.1 to 8.3 kg for Africa
1 Oil Crop Breeding and Genetics 3
(FAOSTAT 2007). Subsequently, the projected rise in vegetable oil consump-

tion by 30% during the decade from 2005 to 2015 will be caused by increases of
per caput food oil consumption in China, India and Latin American coun-
tries, whereas in the European Union and North America it will be driven by
the strongly growing demand for bio-fuels (Thoenes 2006).
The largest increases in world average yield were found in oil palm and
rapeseed (Table 1.1), which can only partly be attributed to plant breeding,
as for both crops the expansion in planting area occurred predominantly in
highly productive environments, i.e. Indonesia and Malaysia for oil palm,
and Europe for rapeseed. Nevertheless, plant breeding has undoubtedly
played a key role in production increases over the past 25 years: In oil
palm, the introduction of hybrid cultivars derived from crosses between Deli
(thick-shelled dura population) and shell-less pisifera or thin-shelled tenera
populations, reciprocal recurrent selection, and the achievement of homo-
geneous planting populations of a favourable genotype through clonal
micro-propagation of hybrids instead of seed propagation are considered
driving forces of the huge yield increase (Soh et al. 2003). In oilseed rape,
genetic progress is attributable to pure line improvement through enhance-
ment of agronomic features, disease resistance and incorporation of the
doubled-haploid technique, whereas high-yielding hybrid cultivars are gain-
ing momentum only recently (Snowdon et al. 2007).
Generally, oil crop breeding is a more complex undertaking than breed-
ing of cereals or legumes, as most oil crops are dual- or multi-purpose
crops, which requires the simultaneous manipulation of different quality
characters. In soybean, oilseed rape, sunflower and a number of other oil
crops, the protein-rich meal is of economic significance beside the oil.
However, a highly negative correlation between oil and protein content is
a major impediment to breeding progress in these crops. In soybean, aver-
age seed oil content is 20% and protein content is 40% with a long-term
tendency of slight increases in oil and decreases in protein content; as both
constituents are important in international trade, economic models based on
oil and protein prices have been proposed as a selection index in breeding of
high value soybeans (Leffel 1990). In oilseed rape, selection of strains
exhibiting the yellow seed color character, which is associated with a thinner
seed coat and lower fibre content than in black-seeded genotypes could be a
strategy of simultaneous improvement of both oil and protein content
(Badani et al. 2006b). In cotton, fibre yield and fibre quality are the main
crop features, whereas cotton seed oil is a by-product and therefore oil
content is not the major breeding objective. In linseed or flax, there are
two main morphotypes of cultivars for either oil production from seed
(linseed) or bast fibre production from stems (fibre flax), whereas dual-
purpose cultivars are rare, and production of both high quality seed and
fibre from the same crop is difficult agronomically. Only recently, the
utilisation of short-fibre linseed straw is discussed for applications in the
emerging field of non-woven materials, and selection criteria for breeding of
dual-purpose linseed cultivars have occasionally been suggested (Rennebaum

et al. 2002; Foster et al. 2000). Moreover, issues such as the specific
requirements of oilseed quality analytics, crop product diversification, the
handling of cytoplasmic male sterility in hybrid crops with hermaphroditic
flowering, and the introduction of genetically engineered cultivars or traits
add to the complexity of oil crop breeding.
Earlier reviews of oil crop breeding have focused on major breeding objec-
tives (Knowles 1983), on breeding methods (Knowles 1989) or on the repro-
ductive systems of oil crop species which determine both the breeding strategy
applicable and the resulting type of cultivar (Arthur 1994). More recently,
excellent reviews have been published on breeding for specific fatty acid
composition (Burton et al. 2004), on the different aspects of improving oil
quality (Velasco and Fernández-Martı́nez 2002), and on genetic engineering
the pathways of oil biosynthesis (Dyer and Mullen 2005). This review
addresses two key features of present day oil crop breeding, genetic diversity
and oil content; the emphasis will be put on annual oilseeds rather than on
perennial crops.
1.2 Domestication and Genetic Diversity

Domestication is an evolutionary process of genetic development, in which
natural selection is replaced by human selection shaping crop plants for specific
needs. Typical changes occurring during the development from a wild plant to a
domesticated crop are referred to as the domestication syndrome; they include
the loss of seed dormancy, increased rates of self-pollination, adoption of
vegetative propagation, increase in yield of seed or other plant organs utilized,
compact growth habit, loss of seed dispersal, increase in number and size of
seeds and inflorescences, changes in color, taste and texture, and decrease in the
content of toxic substances (Gepts 2002). Other important changes include the
alteration of photoperiod sensitivity, adaptation to agricultural soils and agro-
nomic treatments, and the adaptation to new environments often far away from
the center of origin.
Cereals, legumes and fruits were among the first crop plants utilized by
mankind; domestication of cereals dates back some 12 000 years and is
considered as the decisive impetus of Neolithic revolution, the transition
from a hunting and gathering lifestyle to a sedentary agriculture-based
society (Salamini et al. 2002). Oil plants were not among those first
crops domesticated, most of them appeared much later in history, as
their utilization and handling requires specific knowledge and techniques
not available to early agriculturalists. The comparatively late appearance
of major oil crops does have consequences on their status of domestica-
tion, on the development of genetic diversity, and subsequently on avail-
ability of germplasm resources.
1.2.1 Domestication of Oil Crops
The domestication status of oil crops is fairly divergent depending on their

agricultural history. While few oil crops are fully domesticated, many others
express various wild type characteristics, as illustrated in some prominent
examples: Seed dormancy is still a problematic feature of sunflower which
disallows rapid germination of lost seed, but instead causes volunteer sun-
flowers in the following season; pod dehiscence and subsequent seed shattering
may cause considerable yield losses in soybean, oilseed rape, sesame and other
oilseeds; self-pollination is prohibited in several oilseed brassicas due to self
incompatibility; anti-nutritional factors such as protease inhibitors are present
in soybean, oleuropein, a bitter phenolic compound is found in olive; and toxic
components such as glucosinolates in oilseed brassicas or gossypol in cotton
have only been reduced recently. In addition, new oil crops only grown for their
unique fatty acid patterns, such as lesquerella, crambe, cuphea, meadowfoam
or jojoba exhibit numerous wild type characteristics apart from poor
productivity.
Flax or linseed (Linum usitatissimum L.) is today considered to be the oldest
oilseed in the world having been domesticated in the Near East region 10 000
years ago and serving as a source of both oil and fibre from prehistoric time
until present (Allaby et al. 2005). It has been under discussion whether oil or
fibre was the primary reason of domestication, and whether domestication took
place once or happened several times in independent domestication events in
different diversity regions of flax (Diederichsen and Hammer 1995). New
evidence from network analysis of genetic diversity in the stearic acid desaturase
locus sad2 suggests a single domestication event of cultivated flax from its wild
progenitor Linum angustifolium Huds.; moreover, an oilseed type of flax is
proposed as the first domesticate, while fibre flax appears as a later descendant
from oilseed flax (Allaby et al. 2005).
Sesame (Sesamum indicum L.) has often erroneously been described as the
oldest oilseed in human use with a probable origin in Africa, as sesame is a
historically and culturally important crop plant, and there is a high diversity of
Sesamum species on the African continent (Bedigian 2003). However, clear
evidence from archeology, history as well as from botanical, chemical and
genetic data suggests that sesame has been domesticated on the Indian sub-
continent during the period from 3050 to 3500 BC, and that S. malabaricum
Burm., a wild sesame species occurring in India exclusively is the progenitor of
cultivated sesame (Bedigian 1998, 2003).
Sunflower (Helianthus annuus L.) was domesticated by Native North Amer-
icans about 4, 300 years ago from wild H. annuus in the now east-central United
States (Wills and Burke 2006); in addition, multiple evidence for an indepentent
domestication event in Mexico has also been presented (Lentz et al. 2008).
Sunflower was then utilized as a multi-purpose crop, but became an oilseed
only in the late 18th and early 19th century in Russia, from where it spread over
Europe and was later re-introduced from Russia to North America as an oilseed
crop (Putt 1997). From the cross between a cultivated and a wild sunflower
genotype with subsequent QTL analysis, Burke et al. (2002) gained insight into
the genetics of sunflower domestication: Only a few major QTL were found, the
two strongest QTL affected the number of selfed seeds (self-compatibility);
moreover, selection for increased achene size was an important feature of sun-
flower domestication, a high frequency of favourable alleles was present in wild
sunflower, and a majority of sunflower domestication traits was non-recessive.
Soybean (Glycine max (L.) Merr.) was domesticated from the wild Glycine
soja Sieb. & Zucc. in the northeast of China (Manchuria) in the period
1500–1100 BC (Hymowitz 2004) probably in multiple domestication events,
as suggested by chloroplast DNA diversity between wild and cultivated soy-
beans (Xu et al. 2002). So, despite the popular myth claiming soybean to be one
of the oldest crops utilised by mankind (Hymowitz and Shurtleff 2005), it is a
comparatively young crop plant. And much later, during the North Song
Dynasty (960–1127) soybean was recognized as a source of vegetable oil
(Huan and Bao 1993).
Oilseed rape (Brassica napus L.) is known only since the 13th century as an oil
crop (Snowdon et al. 2007; Downey and Röbbelen 1989). As an amphidiploid
interspecific hybrid and probably with a polyphyletic base (Song et al. 1988) it
originated in the Mediterranean region of southwest Europe, where the two
diploid parental species B. oleracea L. (cabbage) and B. rapa L. (turnip) overlap
in their natural habitats. Apart from oilseed rape, the species Brassica napus is
comprised of related forage and vegetable forms (e.g. Soengas et al. 2006), but no
true wild forms are known, which also underlines the recent origin of this species.
1.2.2 Oil Crop Germplasm
The availability of germplasm with sufficient genetic diversity is essential for a

continuous breeding progress. Jones (1983) emphasized the particular need of
preserving oil crop germplasm, as almost all of the major oil crops are now
cultivated far away from their primary centers of origin. Therefore, they do
have a comparatively narrow genetic base classically made up by relatively few
plant introductions who represent the ancestors, from which elite germplasm
and further breeding material is developed.
As shown above, most oil crops gained economic importance during the last
couple of decades only, and many of them are very young crops in terms of their
cultivation and utilisation history as oil plants. These appear to be the main
reasons why oil crops are poorly represented in ex situ germplasm collections at
present. In Table 1.2, a summary is presented on numbers of accessions for oil
crops versus other crops held by the three major genebank associations, which
represent the most significant institutions conserving genetic resources. For all
three associations, cereals such as Triticum sp. (mainly bread and durum
Table 1.2 Accession numbers of crops in general and oil crops in three major genebank
associations (ex situ collections)
Genebank association Crops in general Accessions Oil crops Accessions
CGIAR centers Triticum sp. 114, 721 Soybean1 15, 904
(SINGER) Rice 111, 303 Peanut 14, 694
Sorghum 36, 805
Barley 38, 067
Maize 25, 827
Chickpea 30, 063
Lentil 10, 733
CGIAR total 689, 578
EURISCO Triticum sp. 156, 045 Linseed/flax 17, 226
European Plant Barley 75, 033 Soybean 11, 408
Genetic Maize 42, 267 Oilseed rape 4, 879
Resources Oat 23, 149 Sunflower 4, 444
Search Rye 10, 254 Poppy 4, 114
Catalogue Sorghum 6, 234 Peanut 2, 575
Common bean 30, 845 Cotton 1, 957
Pea 24, 767 Sesame 1, 661
Lentil 5, 635 Safflower 728
Faba bean 5, 600 Olive 421
EURISCO total 1, 000, 175
USDA National Triticum sp. 55, 942 Soybean 19, 277
Plant Germplasm Barley 28, 438 Peanut 6, 831
System Sorghum 42, 666 Cotton 5, 794
Corn 25, 468 Linseed/flax 2, 863
Oat 21, 837 Sunflower 2, 759
Rice 19, 470 Safflower 2, 373
Phaseolus sp. 14, 928 Sesame 1, 226
Chickpea 6, 019 Castor 1, 043
USDA total 477, 077
1
World Vegetable Center (AVRDC, Taiwan, as part of SINGER network).
Sources: CGIAR: http://www.singer.cgiar.org/, 30 April 2007
EURISCO: http://eurisco.ecpgr.org/, 30 April 2007
USDA: http://www.ars-grin.gov/npgs/stats/, 30 April 2007
wheat), rice, barley or sorghum and legumes such as chickpea, pea, lentil or
phaseolus beans have been conserved in clearly higher numbers than oil crop
species: The genebanks of the international agricultural research centers
(CGIAR group, SINGER network) hold a significant peanut collection and a
partly vegetable type soybean collection, but generally oil crops are not on their
list of mandate crops. The European national germplasm collections, linked
together in EURISCO, an internet germplasm search catalogue, hold signifi-
cant numbers of linseed/flax and soybean accessions; for oilseed rape and
sunflower, the two most important European oil crops, accession numbers
are much lower and in the same magnitude as for poppy, which is of very
regional importance only. The United States National Plant Germplasm
System holds significant collections of soybean, peanut and cotton accessions in

their genebanks, which represent the major US oil crop species. In addition to
the accessions listed in Table 1.2, important oil crop germplasm is also main-
tained by institutions in Canada, Argentine, Brazil, China, India, Australia and
few other countries.
Generally, the number of accessions per species held in ex situ collections is
an indicator of past collection activities and the availability of germplasm, but
not a sound measure of genetic diversity. For the accessions of linseed/flax,
Diederichsen (2007) reviewed the ex situ collections world-wide: More than 46,
500 accessions of linseed are present in at least 33 public genebanks; however,
based on analyses of duplications, the author estimates that only 10–15,000
accessions are unique. In soybean, more than 170,000 accessions are maintained
in genebanks, out of which more than two thirds are duplications and about
45,000 are considered unique genotypes (Carter et al. 2004).
Although present in lower number than cereals and legumes, oilseeds such as
linseed, soybean and peanut appear to be well represented in ex situ collections,
while germplasm availability of minor and new oil crops is very limited
(Thompson et al. 1992), and therefore enhancing germplasm collections of
these species will be an important activity ensuring future breeding progress.
1.2.3 Genetic Diversity in Oil Crops – Selected Examples
An overview of the genetic diversity present in the primary and further gene-
pools of a given species is of great interest both to plant breeding and conserva-
tion management. Technically, estimates of genetic relationship may be
obtained from pedigree information, phenotypic data, or molecular poly-
morphisms on the protein or DNA level, and by applying an appropriate
measure of genetic distance (Mohammadi and Prasanna 2003). In oil crops,
various conclusions for plant breeding have been drawn from analyses of
genetic diversity for particular species and populations, as outlined in selected
examples from soybean and oilseed rape.
Soybean genetic diversity has meticulously been investigated from various
points of view and was reviewed by Carter et al. (2004). Pedigree analysis and
calculation of coefficients of parentage revealed that the genetic base of North
American soybean cultivars is narrow as compared to Asian soybeans: While
only 26 ancestors contributed 90% of genes to 258 public cultivars in North
America (Gizlice et al. 1994), it is more than 339 ancestors which contributed
90% of genes to 651 Chinese soybean cultivars (Cui et al. 2000) and more than
74 ancestors which contributed 90% to 86 modern public Japanese cultivars
(Zhou et al. 2000).
Using RAPD markers, Li and Nelson (2001) found a larger genetic diversity
in Chinese accessions than in Japanese or South Korean accessions and were
able to clearly separate Chinese soybeans and those from Japan or South
Korea, respectively. In a diversity study based on AFLP markers, Ude et al.

(2003) suggested to utilize Japanese elite cultivars in order to widen the narrow
genetic base of North American soybeans, as they are more distinct from North
American cultivars than Chinese ones. In numerous other studies, molecular
markers were used to investigate special issues such as variation in vegetable
soybeans (Mimura et al. 2007) or diversity between cultivated and wild soybean
accessions and their geographical genetic differentiation (Chen and Nelson
2004; Xu and Gai 2003).
The phenotypic diversity determined for 15 traits of over 20,000 soy-
bean accessions from the Chinese national soybean collection is represent-
ing a highly valuable information pool for breeding and has further been
used to propose a single geographical center of soybean diversity down-
stream the Yellow River Valley (Dong et al. 2004). Phenotypic data from
25 leaf, stem and seed composition traits of North American and Chinese
soybean cultivars have also been utilized to verify the narrow genetic base
of North American soybeans, which probably represents a subset of the
wider genetic base of Chinese cultivars (Cui et al. 2001); phenotypic dis-
tinctness of these two genetic pools is considered to be the result of
continuous selection for adaptation to contrasting environmental condi-
tions, which now offers new opportunities for reciprocal broadening the
genetic bases by introducing exotic parents.
Marker-assisted introgression of genes from exotic or wild sources through
backcrossing is occasionally considered as enhancing the genetic base of soy-
bean (Lee et al. 2007). However, while backcrossing may bring in the beneficial
effect of a particular allele into adapted breeding material, it does not enlarge
the overall genetic base (Carter et al. 2004); otherwise, backcrossing the geneti-
cally engineered tolerance to the herbicide glyphosate into many commercial
soybean cultivars also did not reduce the genetic base of North American
soybean cultivars (Sneller 2003).
In oilseed rape, genetic diversity is considered to be low because of the short
cropping history and the strong breeding focus on seed quality characters, i.e.
low erucic acid and low glucosinolate contents which narrowed down the
genetic base. Therefore, artificial resynthesis of oilseed rape from its diploid
progenitors cabbage and turnip is practised in order to broaden the genetic base
of oilseed rape (Becker et al. 1995; Seyis et al. 2003; Basunanda et al. 2007),
although resynthesized rapeseed lines exhibit a low yield potential and inferior
seed quality. Resynthesis has repeatedly been used for gene introgression into
cultivars, e.g. for various disease resistances or yellow seed color (Snowdon
et al. 2007). Apart from resynthesis, enriching the genetic base of oilseed rape
has been suggested by hybridizing European and Chinese elite oilseed rape lines
(Hu et al. 2007), or by utilizing diversity existing in vegetable or fodder crop
types of Brassica napus, despite their inferior oil and meal quality (Hasan et al.
2006).
Due to the present transition from pure line breeding to hybrid breed-
ing, genetic diversity in oilseed rape is receiving new attention, as heterotic
pools of accessions with sufficiently large genetic distance need to be

formed for maximum hybrid performance (Snowdon et al. 2007). Signifi-
cant relationships between parental genetic distance and hybrid oilseed
rape performance have been described (Diers et al. 1996; Riaz et al.
2001; Shen et al. 2006), but were considered not sufficient for prediction
of heterosis. For improvement of hybrid performance, Quijada et al.
(2004) suggested the introgression of European winter oilseed rape geno-
mic segments into Canadian spring canola, as superior hybrid performance
was found in testcrosses between these two genepools. A different strategy
for increasing hybrid performance of oilseed rape has been proposed by Li
et al. (2006a), who found considerable heterosis in crosses between natural
Brassica napus parents and a new type of Brassica napus containing the A
subgenome of B. rapa and the C subgenome of B. carinata thus realizing
intersubgenomic heterosis.
1.3 Recent Milestones in Oil Crop Breeding

Over the past few decades, breeding research in oil crops has seen a number of
crucial results which had significant impacts on the subsequent development
of world-wide oil crop production (Table 1.3). Improvement of both oil and
meal in oilseed rape by reducing erucic acid content of oil (canola quality) and
glucosinolate content of meal are two most prominent milestones contributing
to the expansion of world oilseed rape acreage from less than 10 million
hectares in the early 1970s to more than 27 million hectares in 2005 (FAO-
STAT 2007). Moreover, high oleic (Schierholt et al. 2001) and low linolenic
(Rücker and Röbbelen 1996) oilseed rape represent further improvements of
nutritional value and oxidative stability. Relevant changes in fatty acid com-
position have also been achieved in sunflower, soybean and linseed (Table
1.3). Additional examples of alterations in fatty acid composition for parti-
cular crops have been summarized by Velasco and Fernández-Martı́nez
(2002). In sunflower and oilseed rape, cytoplasmic male sterility (cms) allowed
for the development of hybrid cultivars (Table 1.3), whereas in oil palm hybrid
breeding and micropropagation of planting material have contributed to the
success of that crop (Basri et al. 2005). Other biotechnologies such as the
production of doubled haploids in rapeseed (Chen et al. 1994) helped to
accelerate the breeding progress. The perhaps most prominent examples of
genetic engineering and molecular genetics in oilseeds are glyphosate tolerant
soybean and the integrated soybean linkage map (Table 1.3), but genetic
engineering also has a significant impact in oilseed rape (herbicide tolerance,
engineering fatty acid biosynthesis pathways; Snowdon et al. 2007) and in
cotton (Bacillus thuringiensis toxin mediated insect resistance; Christou et al.
2006) at present.
Table 1.3 Recent milestones in oil crop breeding with relevance to world oil crop production
Milestone Year Comment References
Low erucic acid 1959 Character found in German spring-type rapeseed cv. Liho, allowed Stefansson et al. (1961)
rapeseed for the development of high oil quality rapeseed cultivars and
subsequent conversion to ‘canola’ quality cultivars in Canada and
Europe in the 1970s
Low glucosinolate 1968 Found in the Polish spring-type rapeseed Bronowski, lead to release of Josefsson and Appelqvist (1968)
rapeseed low glucosinolate meal cultivars from the 1970s on (00-quality and Lein (1970)
cultivars)
Cms sunflower 1968 Source of cms from interspecific cross Helianthus petiolaris x Leclercq (1969)
H. annuus, introduction of hybrid cultivars in the 1970s
Cms rapeseed 1968 Cytoplasmic male sterility found in Japanese radish (Ogura-cms), Ogura (1968) and Fu (1981)
1 Oil Crop Breeding and Genetics
later transferred to Brassica napus in France; cms in Chinese

rapeseed cv. Polima; at present the two major cms sources for
hybrid seed production in oilseed rape
High oleic sunflower 1976 Induced mutant in sunflower variety VNIIMK 8 931, base of cultivar Soldatov (1976) and Burton et al.
Pervenets, from which high oleic sunflower lines with over 90% (2004)
oleic acid and high oleic hybrids were developed
Low linolenic acid 1986 Two induced mutants from cv. Glenelg with a combined linolenic acid Green (1986)
linseed content below 2% for linseed oil with improved oxidative stability
Low linolenic soybean 1986 Three induced mutant allels with reduced linolenic acid content Fehr et al. (1992) and Ross et al.
combined together for a 1% linolenic acid soybean oil with better (2000)
stability and less trans fatty acid formation
Glyphosate tolerant 1996 Genetically engineered soybean (Roundup Ready soybean) containing Padgette et al. (1995)
soybean EPSP synthase from Agrobacterium sp. with tolerance to the
herbicide glyphosate, first commercially grown in 1996, reached
over 50 million hectares in 2005
Integrated soybean 1999–2004 Integrated high-density soybean linkage map with more than 1800 Song et al. (2004)
linkage map genetic markers (mainly SSRs), useful in fine-mapping genes, map-
based cloning or QTL analysis
11
1.4 Specific Breeding Objectives
Apart from agronomic performance and resistances, oil content of seed or fruit
is the breeding objective economically most important to growers and primary
processors. While breeding for oil quality, i.e. fatty acid composition, has been
the subject of earlier reviews (e.g. Velasco and Fernández-Martı́nez 2002) and is
being dealed with in dedicated crop chapters, various aspects of oil content will
be presented here. Additionally, newly arising breeding objectives of altering
seed composition for health and industrial applications will as well be covered
within the present section.
1.4.1 Oil Content

1.4.1.1 Oil Bodies and the Cytology of Oil Content
Most storage lipids of oilseeds are composed of triacylglycerols which are
synthesized during seed filling. De-novo biosynthesis of fatty acids has been
well presented in earlier reviews (e.g. Stumpf 1989; Harwood and Page 1994),
whereas newer reviews also cover the potentials of genetic engineering fatty acid
synthesis in oil plants (Dyer and Mullen 2005; Napier 2007; Singh et al. 2005).
Fatty acid synthesis is located in plastids of cells in developing embryos, from
where fatty acids activated with coenzyme A are released and accumulate in a
compartment formed by layers of the endoplasmatic reticulum. Inside the
endoplasmatic reticulum, fatty acids may undergo different modifications and
finally are esterified to form triacylglycerols. Due to their hydrophobic nature,
the accumulation of triacylglycerols results in bulges of the endoplasmatic
reticulum from where oil bodies (oleosomes) are developing (Dyer and Mullen
2005) which are the microscopically visible oil bearing structures in mature
seeds. Wältermann and Steinbüchel (2005) have illustrated the most widely
accepted model of oil body formation in oilseeds (Fig. 1.2). From a bulge
formed by triacylglycerols, an oil body is developing and surrounded by a
monolayer of phospholipids, which is derived from the outer leaflet of the
endoplasmatic reticulum. Subsequently, oleosine protein units are embedded
in the phospholipid layer, and the oil body is separating from the endoplasmatic
reticulum. The central domain of the oleosine protein is hydrophobic and
therefore contacting the lipid matrix, whereas both termini are directed towards
the cytoplasm. Oleosine proteins are present in all oilcrops with seeds under-
going dehydration during seed maturation but are not found in oil bodies of
non-desiccating species such as olive, avocado or other tropical oil plants
(Murphy and Vance 1999; Wältermann and Steinbüchel 2005). The size of
oil bodies is dependent on the plant family; the diameter of oil bodies is
between 0.3 and 0.8 mm in Brassicaceae oilseeds, between 0.5 and 2.0 mm in
cotton, linseed and maize, and often above 2 mm in poppy, sunflower and
sesame (Menge and Seehuber 1988; Tzen et al. 1993; Mantese et al. 2006);
Fig. 1.2 Model of oil body development in oilseeds (from: Wältermann and Steinbüchel 2005;
image kindly provided by the authors and used with permission from the American Society for
Microbiology)
very large oil bodies (5–50 mm in diameter) are found in non-desiccating

species (Murphy and Vance 1999). Oleosines regulate the size of oil bodies,
they provide stability during desiccation and rehydration (Peng et al. 2003;
Murphy and Vance 1999) and might be a target to genetic modification of
lipid accumulation (Siloto et al. 2006) and subsequently oil content.
1.4.1.2 Botanical Features of Oil Content

Storage lipids are synthesized, stored and later re-metabolized in the same
tissues within seeds or fruits, as they cannot be translocated within a plant
because of their hydrophobic nature. As storage lipids are a seedlings major
source of energy during germination and emergence, oil bodies are concen-
trated in embryonic tissues, i.e. parenchymatic cells of cotyledons and the
embryo axis in oilseeds, or in the embryo (mainly the scutellum) of cereals,
whereas endosperm tissue is devoid of storage lipids except for castor and few
other species. The basis of genetic variation in oil content may therefore be
variation in size or density of oil bodies, or variation in the proportion of
embryonic tissue containing storage lipids relative to total seed or fruit mass
which is most relevant in practical breeding for high oil content.
In sunflower, Mantese et al. (2006) investigated the temporal and histologi-
cal patterns of lipid accumulation in genotypes with achene oil content ranging
from 300–330 g/kg (low oil content) up to 450–550 g/kg (high oil content). They
reported a tendency of a slightly larger oil body diameter in high oil content
genotypes as compared to a low oil content genotype. While absolute oil mass
of embryo was similar in high and low oil content genotypes, embryos of low oil
genotypes were larger and thus had a lower density of oil bodies; moreover, in
cotyledon transsections of low oil genotypes a significantly larger cell area was
occupied by protein bodies than in high oil genotypes.
While variation in size and density of oil bodies would contribute to the
increase of oil content in small increments, major steps towards improvement of
oil content have been achieved in many oilseeds through selection for reduced
pericarp or thin testa mutants, as shown in Fig. 1.3 for sunflower, rapeseed,
linseed, poppy and oil pumpkin, respectively.
In sunflower (Fig. 1.3A), confectionary genotypes have large achenes with a
thick pericarp (hull) and an oil content of 200–300 g/kg, whereas oilseed
Fig. 1.3 Low (left) and high

(right) oil content accessions
of sunflower (A), oilseed
rape (B), linseed (C), poppy
(D) and oil pumpkin (E),
respectively, differing in
testa or pericarp thickness
cultivars have small achenes with thinner pericarp and an oil content of
400–550 g/kg (Mantese et al. 2006; Tang et al. 2006). It has been estimated
that prior to 1970 two-thirds of increase in sunflower oil content resulted from a
reduction of hull percentage and one-third from increases in kernel (seed) oil
content, while over the last decades almost all of the increase in achene oil
content has been achieved through an increase in kernel oil content (Miller and
Fick 1997). In sunflower cultivars released in Argentine from 1930 to 1995,
achene oil content was increased from 350 to 550 g/kg, while the ratio of kernel
to achene weight was increased from 0.6 to 0.8 by selection for high oil content
(López Pereira et al. 2000). In safflower, another Asteraceae oil crop, increases
in oil content from 390 to 470 g/kg were similarly achieved through selection of
achenes with thinner hulls (Knowles 1983).
In oilseed rape (Fig. 1.3B), the yellow seed character was introduced
through resynthesis from a Brassica rapa source (Liu et al. 2005). The oil
content of yellow-seeded lines with thin testa is 445 g/kg, whereas in compar-
able lines with black seeds an oil content of 399 g/kg has been reported
(Badani et al. 2006b). Moreover, a major QTL for yellow seed colour was
located in the same position as a QTL for reduced fibre content in different
populations, and candidate genes for the yellow seed coat character have been
proposed from the flavonoid biosynthesis pathway (Badani et al. 2006a), as
the black seed colour of oilseed rape is made up by proanthocyanidins (con-
densed tannins).
In linseed, the yellow seed character (Fig. 1.3C) is known to be associated
with higher oil content and larger seed weight, but has shown agronomic
disadvantages such as a lower seed germination rate due to seed injury and a
higher incidence of root rot (Culbertson et al. 1960; Bradley et al. 2007). In a
large linseed germplasm collection, yellow seed is also associated with a
significantly higher oil content and a larger seed weight than brown seed
(Diederichsen and Raney 2006). The yellow seed character is controlled by
several independent loci (Mittapalli and Rowland 2003), but causal relations
for the association between yellow seed and high oil content are missing.
Similar to other oilseeds, yellow seed colour in linseed may be due to a thinner
seed coat of yellow seeds as compared to brown seeds. This view is also
supported by the finding that linseed mucilage, which consists of polysacchar-
ides located in the epidermis of the seed coat, is present in lower concentra-
tions in yellow-seeded accessions than in brown-seeded ones (Diederichsen et al.
2006). Similarly, yellow or white seeds of poppy (Fig. 1.3D) are superior in oil
content to blue seeds (Azcan et al. 2004; Bernáth 1998), which may as well be due
to a thinner seed coat. In oil pumpkin (Fig. 1.3E), a mutation prohibiting the
lignification of testa (hull) gave rise to utilising pumpkin as an oilseed crop
instead as a vegetable in some Central European countries. Oil content of seeds
with thick, lignified testa is below 300 g/kg, whereas oil content of cultivars
utilizing the thin testa mutation (Styrian oil pumpkin cultivars) is between 350
and 500 g/kg (Idouraine et al. 1996; Murkovic et al. 1996). Lignification is
controlled by one major gene with the thin testa character being recessive, and
several minor genes may cause partial lignification of thin testa genotypes (Tepp-
ner 2004; Zraidi et al. 2003).
In maize, 100 generations of selection for either high or low oil content
yielded strains with an oil content of above 20% or below 1%, respectively, in
the Illinois long-term selection experiment for oil and protein (Dudley and
Lambert 2004). As the oil-bearing tissue of a maize seed is the embryo, high
oil content was associated with an enlargement of the scutellum (Moose et al.
2004). In another maize synthetic (Lambert et al. 2004), high oil content was
associated with a reduced starch content, whereas protein content remained
unaffected; moreover, seed weight was reduced, and the proportion of the
embryo was increased, while endosperm was decreased through selection for
high oil content.
As cotyledons hold the oil-bearing tissues in most of the annual oil-
seeds, selection for cotyledon size, as demonstrated for Brassica rapa (Tel-
Zur and Goldman 2007), might be an alternative route to improve seed oil
content.
1.4.1.3 Genetics of Oil Content

Oil content is a quantitative character controlled by both the genetics of a
cultivar and the environment. While the genetics of particular fatty acids had
been well characterised in many oil crops, information about the inheritance of
oil content was meagre for a long time. As an example, oil content of rapeseed
was assumed to be controlled by mainly additive gene action with dominance
being not significant and epistasis absent, and the number of genes involved in
oil content was estimated to be lower than that for seed yield (Röbbelen and
Thies 1980). During the last decade, however, mapping of quantitative trait loci
(QTL) has brought more insight into the genetics of oil content and the func-
tional principles behind particular QTL.
In soybean, 69 QTL for oil content were listed in the USDA SoyBase database
(http://soybeanbreederstoolbox.org, October 2007) with most listings occurring
for linkage groups A1, E, I and L. While most putative QTL do have small effects
on oil content, one to four major QTL explain more than 10% of the phenotypic
variation in different segregating populations (Lee et al. 2007). Three to eight QTL
affecting oil content have also been reported for oilseed rape (Ecke et al. 1995;
Burns et al. 2003) and other cruciferous oil crops such as Brassica juncea
(Mahmood et al. 2006) or Camelina sativa (Gehringer et al. 2006), while one or
two of up to 14 QTL explained more than 10% of oil content variation in oilseed
rape (Delourme et al. 2006). Additive gene action has been proposed as the way of
action for most QTL influencing oil content. In addition, Leon et al. (2003)
reported dominant QTL effects for oil content in a bi-parental sunflower popula-
tion. Digenic epistasis in oil content QTL has been described for oilseed rape
(Zhao et al. 2005; Delourme et al. 2006), Brassica juncea (Mahmood et al. 2006),
soybean (Lark et al. 1994), or oat (Zhu et al. 2004). In the maize long-term
selection for oil and protein, large numbers of epistatically acting QTL for oil,
protein and starch content suggest the importance of epistasis for continuing
selection response, particularly at lower levels of genetic variability (Dudley
2008).
The negative correlation between seed oil and protein content known
from various oilseeds is also evident on the QTL level. QTL regions
associated with oil content are frequently found to control protein as
well and vice versa. This association may be due to tight linkage between
oil and protein alleles in repulsion phase or to pleiotropic effects. In
soybean, Lee et al. (2007) estimated that about 58% of oil content QTL
are also associated with protein across a number of studies. Chung et al.
(2003) described a QTL affecting oil content, protein content and grain
yield of soybean and proposed the presence of a single QTL pleiotropically
affecting both oil and protein. Nichols et al. (2006) narrowed down an oil and
protein QTL region on soybean linkage group I to a 3-cM marker interval by
fine-mapping, but still could not discriminate between either pleiotropy or
close linkage of two loci affecting both traits. In oilseed rape, conditional
mapping of QTL for oil content (Zhao et al. 2006) allowed for discriminating
between true oil or protein QTL as well as the identification of additional oil
QTL as compared to unconditional mapping.
Knowledge on the functional genetic mechanisms and regulatory metabolic
factors controlling oil content is rather limited. In Brassicaceae, genes coding
for erucic acid content may be considered ‘candidate genes’ for oil content,
because the increased chain length and molecular weight of the erucic acid
molecule (C22-body) as compared to C18-fatty acids causes an increase of
total oil content. In oilseed rape, two QTL affecting oil content were at the
same time associated with the two genes for erucic acid content (Ecke et al.
1995) in a cross between a high and a low erucic acid line. Subsequently, these
genes were characterised as fatty acid elongase genes homologous to the Arabi-
dopsis FAE1 gene coding for the elongation of C18:1 (oleic acid) to C22:1
(erucic acid) in oilseed rape (Fourmann et al. 1998) and in Brassica juncea
(Gupta et al. 2004). Contrary to erucic acid, palmitic acid (C16:0) has a lower
molecular weight than C18-fatty acids, which may partly explain its negative
correlation with oil content in oilseed rape, sunflower or soybean (Möllers and
Schierholt 2002; Velasco et al. 2007; Hartmann et al. 1996). Differential gene
expression studies using oilseed rape lines either high or low in oil revealed a
major involvement of genes related to chloroplast function (photosynthesis)
and sucrose metabolism in the expression of oil content (Li et al. 2006b). In oat,
a plastidic acetyl-CoA carboxylase gene which catalyzes the initial step of de
novo fatty acid synthesis was identified as a candidate gene strongly affecting oil
content (Kianian et al. 1999). In an Arabidopsis transformation experiment,
Jako et al. (2001) showed an increase in oil content through over-expression of a
diacylglycerol acyltransferase. The metabolic complexity of oil content as a trait
is also depicted on the proteomics level, where proteins related to energy,
carbohydrate and amino acid metabolism are prominently expressed during
seed filling in oilseed rape and sunflower (Hajduch et al. 2006, 2007), which may
contribute to identifying high level genes regulating oil content.
1.4.1.4 Breeding for Oil Content

Due to the diversity in reproductive systems of different oil crop species,
cultivars can be ascribed to each of the four classical plant breeding categories,
i.e. clones (olive, clonally propagated oil palm), allogamous populations
(coconut, oil palm, castor, outcrossing brassicas), autogamous pure lines
(soybean, linseed, peanut, poppy, sesame), and hybrids (sunflower, oilseed
rape, oil palm, castor). As oil content is mainly controlled by additive gene
action, the transition from open-pollinated or pure line types of cultivars to
hybrids such as in sunflower or oilseed rape did not affect oil content to a great
extent. In oilseed rape, considerable heterosis is present in grain yield, whereas
heterosis is not relevant for oil content (Qian et al. 2007). In soybean, a strictly
autogamous species, heterosis in testcrosses was high for maturity date, plant
height and grain yield, but low for oil and protein content (Lewers et al. 1998).
Thus, the additional gain from hybrid oil crop cultivars is primarily due to
higher grain yield and a subsequent oil yield increase rather than a higher oil
content. In oil palm, contrary to annual oilseeds, not fully inbred parents are
used to produce heterogeneous hybrids; a transition from seed to clonal
propagation through in vitro culture techniques would allow for the multi-
plication of high-yielding individual palm trees thus enhancing oil yield per
unit area (Soh et al. 2003).
Oil crop breeding is a complex undertaking, and selection for oil content
needs to be considered in the context of various other traits and their
biological and economic values. In most breeding populations, the correla-
tion between grain yield and oil content is positive or not significant,
whereas the correlation between grain yield and protein content is negative.
Additionally, correlations between oil content and time to flowering, seed
weight or fatty acid concentrations may also be of relevance, particularly in
recurrent selection for oil content or in trait introgression programs. Oil
content usually appears as quantitative character with medium to high
heritability in most populations, which ensures a significant selection
response.
On the technical level of selection, efficient analytical tools such as
nuclear magnetic resonance (NMR) or near-infrared reflectance spectro-
scopy (NIRS) have been established for non-destructive measurement of
oil content. In addition to the widely used NIRS-based prediction of oil,
protein and moisture content, NIRS calibrations have been developed for
measuring amino and fatty acids (Pazdernik et al. 1997; Velasco et al. 1999;
Kovalenko et al. 2006) and antinutritional components such as glucosino-
lates (Font et al. 2006) or phenolics (Velasco et al. 1998). Moreover, NIRS
procedures for analyzing single seeds for oil content or fatty acid concentra-
tion (Jiang et al. 2007; Tillman et al. 2006; Velasco et al. 1999, 2004) have
been presented. Thus, high-throughput technologies for screening large

numbers of samples for oil content and related quality features on the
basis of individual seeds, individual plants (e.g. Fasoula and Boerma 2005)
or small plots such as hill or single-row plots (Pazdernik et al. 1996) are
available for selection and quality monitoring at various stages of breeding
programs.
1.4.2 Altered Seed Composition for Health and Industrial

Applications
Besides altering the relative amounts of oil and protein in the seed of oil crops,
considerable efforts have been made by oil crop breeders to alter the fatty acid
profile of the oil. There are five major fatty acids found in most of the annual
oilseeds, palmitic, C16:0; stearic, C18:0; oleic, 18:1; linoleic, C18:2; and linolenic
acid, C18:3. Additional fatty acids are found in specific oil crops such as erucic
acid C22:1 in oilseed rape or ricinoleic in castor bean. The manipulation of
relative proportion of the fatty acids has often been practised to address issues
of the healthfulness of oil by reducing the saturated fats (e.g. C16:0) or increas-
ing the levels of monounsaturated fats such as C18:1. Mutagenesis, natural
variation, recurrent selection and genetic engineering have been the approaches
that have been used by oil crop researchers over the past several decades. It is
noted that, although some of these EMS-derived mutations such as the low
linolenic have been developed more than 30 years ago (Rajcan et al. 2005;
Röbbelen and Nitsch 1975; Fehr 2007), many of them have gained in market
importance only recently. The main interest in the low linolenic acid oils (with
less than 3% of C18:3) is due to the lack of need for partial hydrogenation,
which in high C18:3 oils results in the production of the by-product trans-fatty
acids that have been shown as more detrimental to heart health than saturated
fat. The low C18:3 oil only became economically attractive when the Food and
Drug Administration regulated the compulsory food labelling of trans fat
content in the USA, which in turn prompted considerable interest and market
demand by food manufacturers. Similarly, while high oleic sunflower mutants
have been available for a long time, the mid-oleic NuSun sunflower only gained
market popularity when such hybrids developed by the USDA (Miller and Vick
2002) became readily available and a solid customer base had been generated. It
would appear that healthier oils that have been developed as result of mutagen-
esis and selection work by oil crop breeders often have to wait until the market is
ready for them, which sometimes political decisions rather than biological or
health considerations. It is reasonable to expect that other fatty acid profiles
that could address health issues, stability of oil or provide feedstock for specific
food or industrial products will gain market acceptance after years of selection
work by oil crop breeders. Some of the novel profiles that may be of interest in
the future could be a high stearic oil for margarine production, low palmitic
combined with low linolenic for a healthier and more stable oil, high palmitic oil
for biodiesel engines, high polyunsaturates for polyol and other biomaterials
industries, etc.
Numerous clinical studies have been conducted that confirm the health
benefits of non-oil nutritional components of oil crop seeds such as soybean
protein (or peptides as part of it), or nutraceutical components such as gluco-
sinolates of oilseed rape, lignan of linseed, isoflavones and saponins of soybean.
The effects of these compounds include a reduction of heart and coronary
disease, osteoporosis, certain types of cancer, e.g. prostate and breast cancer,
and menopausal symptoms in women. These findings have opened a new
avenue of research for plant breeders and geneticists in their efforts to widen
the array of uses for oil crops and/or their products in support of the nutritional
and nutraceutical industries. Recent efforts by plant breeders have concen-
trated around improving their understanding of the genetic control of the
already recognized functional food components such as isoflavones (Primomo
et al. 2005), tocopherols (Wohleser 2007) or saponins (Rupasinghe et al. 2003).
The options for breeders and industry are plentiful and seemingly limited only
by the type and variation of compounds in the seeds as well as market con-
siderations. Faced with stagnating commodity prices, oil crop breeders are
looking toward nutraceuticals, biobased industrial feedstocks and biofuels as
means to alleviate low and fluctuating profitability in agriculture. The devel-
opment of value-added oil crops and products for the current and emerging
niche markets appears an attractive alternative for breeders and producers.
1.5 Perspectives in Oil Crop Breeding

1.5.1 Technology
On the level of technology, new tools are on the horizon which may affect
strategies and efficiency of oil crop breeding in the near future.
Mutation induction is a well-established technique with special relevance to
oilseed crops: At present, numerous mutants induced and isolated decades ago
are being incorporated in widely grown cultivars to improve their fatty acid
composition and nutritional value (Bhatia et al. 1999); while phenotyping large
populations for a desired mutation is a major bottleneck in mutation breeding
programs, new approaches such as TILLING combine conventional mutation
induction and PCR-based high throughput reverse genetics mutant identifica-
tion (Henikoff et al. 2004; Cooper et al. 2008); thus, numerous new alleles could
be isolated at a given gene locus and phenotyped for their usefulness to breeding
thereafter.
Although genetic markers are widely applied in oil crop breeding at present,
their utilization could be further stimulated by the wider availability of high-
throughput methods, SNP-markers and other techniques. Monitoring of
genetic diversity, prediction of hybrid performance, QTL analysis, marker

assisted introgression and transfer of specific alleles, or marker assisted selec-
tion for traits difficult to phenotype such as disease/pest resistance, seed quality
or health components would undoubtedly increase the efficiency of oil crop
breeding programs.
Other biotechnologies such as doubled-haploids for rapid recovery of
homozygous recombinants or micropropagation of superior planting mate-
rials in perennial crops such as the oil palm are well established techniques
in particular applications. However, while doubled-haploids are widely
used in cereals, oilseed rape and numerous other crops (Forster et al.
2007), species such as the soybean are considered recalcitrant to haploid
production, and almost no research is devoted to that area despite its huge
potential.
Oil crops are the earliest and major group of genetically modified (GM)
crops grown world-wide: The total acreage of GM crops reached over 114
million hectares in 2007 (James 2007) with soybean, maize, cotton and canola
being the lead crops, and herbicide tolerance and insect resistance being the
major traits targeted. Despite of the benefits attributed to biotech crops, con-
sumer acceptance of GM crops and products is low; wider evidence on crop bio-
safety and the development of ‘second generation biotech crops’ (Napier 2007)
with new traits beneficial both to the environment and to consumers as well as
farmers are considered crucial with respect to public opinion on GM crops.
Meanwhile, separate breeding programs are maintained in parallel for GM and
GM-free cultivar development in some crops to serve the different market
requirements.
1.5.2 Biology
On the level of biology, issues such as maintenance of genetic diversity, the

development of new oil crops, stability of quality features in crop harvests, or
adaptation of oil crops to particular growing conditions such as organic farm-
ing may gain importance for future oil crop breeding.
Genetic diversity is comparatively low in most oil crops due to their specific
crop and breeding histories. While this may not be a problem for immediate
breeding work, it could negatively affect the long-term breeding progress,
impede the adaptation to new environments or enhance crop vulnerability
with respect to epidemic pests or diseases. Introgression of wild type alleles by
backcrossing is efficient with respect to a specific target character such as the
protein content of soybean (Sebolt et al. 2000), but will not increase overall
genetic diversity. Wide crosses, recurrent programs, pre-breeding on a wild
species level or hybridizations between wild and domesticated species for trans-
ferring complex characteristics such as the outstandingly high oil content found
in some wild sunflower species (Seiler 2007) are long-term concepts for
increasing diversity while being challenged by the need to maintain an accep-

table level of agronomic performance.
Instead of transferring various traits into few established oil crop species,
development of new crops with specific qualities or the revival of forgotten and
underutilized species represent alternative strategies with the long-term goal of
enhancing agro-biodiversity at large. While various different species of that
kind have been discussed during the past decades, few have appeared on the
crop production and marketing level so far, and considerable research input
may be needed for others.
1.5.3 Utilization
Oil crop utilization perspectives are most difficult to predict due to increasing
market volatility of agricultural commodities in general and a high level of
substitution among oils of different species.
Nevertheless, oil crop breeding for product quality features has been success-
ful in generating diversified markets for vegetable oils. In food utilization of
oils, diversification of products through breeding is constantly producing new
features such as improved shelf life, suitability for frying, reduction of trans-
fatty acid generation during hydrogenation, human blood cholesterol reducing
properties, anti-oxidant and various other health promoting properties. In non-
food utilization, demands from oleochemistry have prompted for the develop-
ment of crops producing long- or mid-chain fatty acids as well as hydroxy and
epoxy fatty acids, while many other properties may be realized by genetic
engineering approaches (e.g. Dyer and Mullen 2005). Demands for energetic
utilization of vegetable oils, e.g. for bio-diesel production, have been less
specific in terms of oil quality so far, and the future position of bio-diesel
production is unpredictable because of the rise of ethanol producing crops as
well as high competition for land due to increasing world market prices for food
and agricultural commodities. Apart from improving oil qualities itself, signifi-
cant plant breeding efforts will also have to be devoted to the wide range of
possible by-products of oil crop production such as protein meals for feeding or
other industrial applications, as successful marketing of by-products has
become a key factor for economic success of oil crop processing.
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Chapter 2
Modifying Vegetable Oils for Food and Non-food
Purposes
Edgar B. Cahoon, Thomas E. Clemente, Howard G. Damude,

and Anthony J. Kinney
2.1 Introduction
Oils and fats are an important source of energy for the human diet and also
contribute significantly to the sensory characteristics of food. Many oils are also
used for non-food applications, although industrial use currently accounts for
only a small proportion of the world vegetable oil production, less than 5% of
total production, mostly for biodiesel. About 80% of edible oils are derived
from plant sources and temperate annual oil seeds (soy, rapeseed, sunflower
and peanut) account for about 60% of this total. Soybean oil is by far the
dominant oil in this category, accounting for over half of the world vegetable oil
production.
Improving the functional and nutritional qualities of vegetable oils has
garnered much attention over the last 15 years or so. This chapter will describe
some of the attempts to genetically improve plant seed oils, with special
emphasis on soybean oil, for food and non-food uses.
2.2 Modulating the Fatty Acid Content of Plant Oils for Food Uses
2.2.1 Fatty Acid Profile and Oil Functionality
The fatty acid profile plays a significant role in both the nutritional properties
and end-use functionality of edible plant oils. With its high percentage of
polyunsaturated fatty acids (>65%), the major food oils derived from seeds
are relatively oxidatively unstable, which limits their utility in food applications.
Historically, oil processors have used partial hydrogenation as a means to
improve upon the oxidative stability of plant oils. This process chemically shifts
the fatty acid profile towards increased saturated and monounsaturated, and
E.B. Cahoon (*)

Center for Plant Science Innovation and Department of Biochemistry, University of
Nebraska, Lincoln, NE, USA
e-mail: ecahoon2@unl.edu

32 E.B. Cahoon et al.
concomitantly reduces the polyunsaturated fatty acids. Partial hydrogenation

also significantly improves solid fat functionality, which is especially useful for
baking and similar applications. This improved functionality comes at a cost to
nutritional quality due to the presence of high concentrations of trans fatty
acids found in partially hydrogenated oils. Trans fatty acids have been corre-
lated with cardiovascular disease (Mensink and Katan 1990; Korver and Katan
2006). Intake of other dietary fatty acids can have a strong influence on overall
health. For example, saturated fatty acids such as palmitic acid, tend to raise
LDL-cholesterol whereas stearic acid has neutral effect (Minihane and Harland
2007). Hence, the development of alternative vegetable oil feed stocks that can
simultaneously improve functionality while maintaining nutritional quality
holds great market potential to both the frying and baking industries.
Commodity soybean oil presently is composed of 14% saturated fatty acids,
18% monounsaturated fatty acid, and 65% polyunsaturated fatty acids.
Through targeted genetic alterations, designer soybean oils are being developed
to address both oxidative stability and nutritional quality. These include oils
high in oleic acid and low in saturated and polyunsaturated fatty acids, oils
elevated in stearic acid, and combinations of reduced polyunsaturate content
with elevated stearic acid.
2.2.2 Conventional Approaches to Fatty Acid Modification
Oleic acid is metabolized to linoleic acid by a single desaturation step carried out
by a D12-desaturase encoded by the FAD2 gene (Heppard et al. 1996). In
soybean there are at least six FAD2 genes that fall into two classes, FAD2-1
and FAD2-2. The FAD2-1 class is primarily embryo-specific, while the FAD2-2
class is generally constitutive, expressed during both vegetative and seed devel-
opmental stages (Tang et al. 2005). Soybean breeders have made great strides to
move an elevated oleic acid phenotype into elite genotypes by exploiting natural
variation in oleic acid levels among various sources of soybean germplasm
(Takagi and Rahman 1996; Rahman et al. 2001; Alt et al. 2005a,b).
Conventional approaches to raise the oleic acid content in soybean oil has led
to the development of ‘‘mid-oleic’’ phenotype, in which seed storage lipids range
in oleic acid from 30 to 70%. The conventional approach to develop the mid-
oleic phenotype has some drawbacks. First, the genetics of the phenotypes
require the stacking of multiple loci (Alt et al. 2005a, b), which may complicate
the breeding process. Secondly, the ‘‘mid-oleic’’ phenotype is affected by envir-
onment, typically requiring growth in warmer climates for stability of the
elevated oleic acid trait to be maintained. This is due to the temperature effect
on the desaturase activity and expression (Heppard et al. 1996; Tang et al. 2005).
The third drawback associated with the conventional breeding approach for a
‘‘mid-oleic’’ soybean is the germplasm that expresses this phenotype is associated
with yield drag (Primomo et al. 2002).
2 Modifying Vegetable Oils for Food and Non-food Purposes 33
In soybean seed the content of palmitate is regulated by a palmitoyl thioes-

terase encoded by FatB genes (Kinney 1997). Low palmitic acid soybean
genotypes have been reported (Bubeck et al. 1989; Primomo et al. 2002). The
low palmitic acid phenotype in soybean has recently been associated with allelic
variation in FatB (Cardinal et al. 2007). Importantly, as the conventionally bred
‘‘mid-oleic’’ soybean, the low palmitic acid genotypes seem to suffer from yield
penalty (Rebetzke et al. 1998; Cardinal and Burton 2007).
The concentration of the cardiovascular ‘‘neutral’’ saturated fatty acid,
stearate, in soybean is controlled by both desaturase (Cheesbrough 1990) and
thioesterase activities (Pantalone et al. 2002). The genetic locus controlling
elevated stearic acid levels in soybean, is designated Fas, and allelic variation
at this locus manifests stearic acid levels ranging from wild type concentrations
of 3%, up to high stearate germplasm with 35% (Bubeck et al. 1989; Rahman
et al. 1995; Pantalone et al. 2002).
Significant amount of progress has been made in developing novel
soybean germplasm, with altered fatty acid profiles, using conventional
breeding strategies. However, as mentioned above there tends to be a yield
drag associated with these improved oil traits. This may be due to the allelic
variants selected for altered fatty acid profile during both vegetative and
embryogenesis development, thereby increasing the probability of a negative
agronomic effect associated with the mutant allele governing the novel oil
phenotype. Moreover, while soybean output traits, such as ‘‘mid-oleic’’, low
palmitic acid, and elevated stearate have value, there are applications in
which stacking of these traits would offer expanded functionality. Given the
genetic complexity regulating the ‘‘mid-oleic’’ trait in soybean, combining
this phenotype with low palmitic and/or elevated stearic acid traits, into elite
soybean genotypes, would be challenging. High oleic acid mutants, with an
oleic content ranging from 60 to 90%, have also been developed in corn,
peanut, canola and sunflower. These mutants all have defective FAD2 genes
(Perez-Vich et al. 2002; Patel et al. 2004; Hu et al. 2006; Belo et al. 2008).
As in soybean, many of these plants have a number of FAD2 genes all
which contribute to seed linoleic acid content (Tang et al. 2005), which
means that the development of mutant lines with useful oleic acid contents
often requires combining several mutant loci (Mikkilineni and Rocheford
2003).
2.2.3 Novel Fatty Acid Profiles in Soybean Derived from the Tools
of Biotechnology
Implementing the tools of biotechnology novel oil traits can be achieved in a

seed-specific fashion, with a single dominant allele. This approach simplifies
breeding and reduces the probability of agronomic performance being
compromised.
Targeted perturbation of FAD2 alleles in a seed-specific fashion in

soybean has been shown to produce a high oleic acid (75–85%) phenotype
in the seed oil (Kinney and Knowlton 1997; Mazur et al. 1999). Modula-
tion of FAD2 expression using this transgenic approach was carried out by
introducing transgenic elements designed to induce post-transcriptional
gene silencing (Cerutti 2003).
High oleic acid soybean derived from down-regulating FAD2, concomitantly
reduces polyunsaturated fatty acids to below 6% (Kinney and Knowlton 1997),
in addition palmitic acid was reduced to approximately 7–8% (a 20% reduction
in palmitate).
For some uses, such as salad oils, it is desirable to reduce saturated fatty
acids, especially palmitic acid as much as possible. It has been shown that the
FATB class of acyl:ACP thioesterases control the release of some saturated
fatty acids, including palmitic, into the cytoplasm, making them available for
oil biosynthesis (Dörmann et al. 2000). Seed-specific silencing of FATB genes
in soybean leads to a major reduction in total saturated fatty acids, from
about 15% to less than 6% (Kinney 1996). When this gene was combined
with a silenced FAD2, oleic acid contents of over 90% were observed (Buhr
et al. 2002). Importantly, field trials with soybean having this novel oil
phenotype, conducted across multiple environments, revealed that the fatty
acid content was not affected by temperature and the agronomic perfor-
mance was not compromised.
For certain food applications, such as baking, solid fat functionality is
needed. This functionality is currently provided by animal fats, palm oil and
hydrogenated vegetables oils, and biotechnology has the potential to provide a
healthy alternative to these fats. It is thought that oils rich in stearic acid could
provide this important function without compromising human health because,
unlike other saturated fatty acids, stearic is not considered to play a significant
role in cardiovascular disease (Mensink 2005; DiRienzo et al. 2008).
High stearic acid vegetable oil has been developed by targeting either down-
regulation of desaturase activity (Liu et al. 2002) or heterologous expression of a
stearoyl-ACP thioesterase (Hawkins and Kridl 2002). Soybeans seeds have sev-
eral D9 desaturase (SAD) genes expressed in their seeds and silencing one of these
genes (SAD3) in a seed-specific manner resulted in soybean oils containing
20–30% stearic acid when compared with just a few percent in commodity
soybean (Booth et al. 2006). The seeds of mangosteen (Garcinia mangostana)
contain stearate as the predominant fatty acid (45–50% of total fatty acids), a
result of a stearate-specific thioesterase. When the mangosteen gene was expressed
in transgenic canola seeds, stearate contents of 20–30% were reported (Hawkins
and Kridl 1998). Similar increases in stearate have been observed when this gene
was expressed in soybean seeds (Kridl 2002).
To replace the solid-fat functionality of many hydrogenated oils the stearate
needs to be a major component of the seed oil, around 30% of the total fatty acids
(Lumor et al. 2007) and germination problems are often seen when the stearic acid
content of seeds is elevated to these levels (Roberts et al. 2006; Clemente
unpublished data). However, by combining the silencing of SAD3 with silencing of

FAD2 in soybean it has been possible to make viable seeds that have an oleic content
in the 50–60% range with stearic around 15–20% (Booth et al. 2002). This combi-
nation of stearic and oleic confers the desired solid fat functionality without
compromising the seed. Improved functionality has also been obtained by combin-
ing increased stearic acid with low linolenic acid oils (DiRienzo et al. 2008). Healthy
oils from these types of seeds have the potential to replace solid fats in a wide range
of baking and heavy-duty frying applications.
The introduction of modified seeds oils such as high oleic acid soybean oil
and high oleic/high stearic oils will go a long way to removing the undesirable
saturated fatty acids and trans fatty acids from the human diet and help
promote cardiovascular health (Korver and Katan 2006; Lichtenstein et al.
2006; Mozaffarian and Willett 2007).
2.3 Next Generation Edible Oils: Producing Long Chain

o-3 Fatty Acids in Seed Oils
2.3.1 Engineering Complex Pathways into Plant Seeds
The efforts toward genetic engineering plants to alter seed oil compositions
described above focused on modifying existing biosynthetic pathways at a
single enzyme step. With advancements in plant transformation and genomic
technologies, expression of significantly more complex gene combinations,
including whole metabolic pathways from heterologous sources, are now
being explored and thus, the flexibility to produce highly functionalized,
higher-value metabolic end-products is also becoming possible. A good exam-
ple of valuable but metabolically complex end products that are of current
significant interest are the long-chain polyunsaturated fatty acids (LCPUFA)
including the o-3 LCPUFA eicosapentaenoic acid (EPA) and docosahexaenoic
acid (DHA). The importance of these fatty acids in human health and nutrition
and current efforts to produce them in oil seed crops will be discussed.
EPA is a 20 carbon fatty acid having 5 double bonds while DHA is a 22
carbon fatty acid having 6 double bonds. Both are considered o-3 fatty acids in
that they have a double bond occurring between the third and fourth carbon
from the methyl end. Arachidonic acid (ARA) is similar to EPA in that it is a 20
carbon fatty acid but lacks the o-3 double bond and the double bond closest to
the methyl end of the fatty acid occurs between the sixth and seventh carbon,
thus making ARA an o-6 fatty acid. Most naturally occurring fatty acids, and
all that are relevant to this discussion, have double bonds having a cis config-
uration. The chemical structure of the fatty acid (i.e. chain length; number,
position and stereochemistry of double bonds) is directly related to its effect on
human physiology including potential health properties and a list of the fatty
acids relevant to this discussion along with their chemical structures is shown in
Table 2.1
Table 2.1 Nomenclature and chemical structures of fatty acids

Carboxyl o
Saturation Common name Abbreviation nomenclature nomenclature
Saturated myristic MA 14:0 14:0
palmitic PA 16:0 16:0
stearic SA 18:0 18:0
Monounsaturated oleic OA 18:1(D9) 18:1 o-9
eicosanoic EA 20:1(D11) 20:1 o-9
Polyunsaturated taxoleic TXA 18:2(D5,9) 18:2 o-9
linoleic LA 18:2(D9,12) 18:2 o-6
g-linolenic GLA 18:3(D6,9,12) 18:3 o-6
pinolenic PNA 18:3(D5,9,12) 18:3b o-6
a-linolenic ALA 18:3(D9,12,15) 18:3 o-3
stearidonic STA 18:4(D6,9,12,15) 18:4 o-3
eicosadienoic EDA 20:2(D11,14) 20:2 o-6
dihomo-g- DGLA 20:3(D8,11,14) 20:3 o-6
linolenic
sciadonic SCI 20:3(D5,11,14) 20:3b o-6
eicosatrienoic ERA 20:3(D11,14,17) 20:3 o-3
arachidonic ARA 20:4(D5,8,11,14) 20:4 o-6
eicosa- ETA 20:4(D8,11,14,17) 20:4 o-3
tetraenoic
juniperonic JUP 20:4(D5,11,14,17) 20:4b o-3
eicosa- EPA 20:5(D5,8,11,14,17) 20:5 o-3
pentaenoic
docosa- DTA 22:4(D7,10,13,16) 22:4 o-6
tetraenoic
docosa- DPAn-6 22:5(D4,7,10,13,16) 22:5 o-6
pentaenoic
docosa- DPA 22:5(D7,10,13,16,19) 22:5 o-3
pentaenoic
docosa- DHA 22:6(D4,7,10,13,16,19) 22:6 o-3
hexaenoic
tetracosa- TPA 24:5(D9,12,15,18,21) 24:5 o-3
pentaenoic
tetracosa- THA 24:6(D6,9,12,15,18,21) 24:6 o-3
hexaenoic
Although the effects of fatty acids on human health may be variable in

different human populations depending on age, sex, race and genetic back-
ground, studies have shown that consumption of fats high in short-chain,
saturated fatty acids leads to increases in low-density lipoprotein (LDL) and
may lead to increased risk of cardiovascular disease (Schaefer 1997). Stearic
acid (SA), an 18 carbon saturated fatty acid, is neutral in its effect on blood
lipids (Kris-Etherton et al. 2005; Davis and Kris-Etherton 2003; Schaefer
1997) and monounsaturated and polyunsaturated fatty acids are inversely
correlated with coronary heart disease (Binkoski et al. 2005;Davis and Kris-
Etherton 2003; Kris-Etherton 1999). Given these trends, reduced consumption
of short-chain, saturated fat and increased consumption of foods rich in

mono- and polyunsaturated fats and oils should lead to an overall healthier
state.
Vegetable oils such as soybean and canola oils are rich sources of poly-
unsaturated fatty acids (o-3 and o-6). The polyunsaturated fatty acids linoleic
acid (LA) and a-linolenic acid (ALA) commonly found in many oilseeds are
not only considered healthy but are, in fact, essential fatty acids since humans
lack the enzymes necessary to produce them. Humans are biosynthetically
capable of producing oleic acid (OA) de novo from glucose or other basic
carbon sources but are incapable of further desaturating OA to LA, and LA to
ALA as they are missing the D12 and D15 desaturase (o-3 desaturase)
enzymes, respectively. Oilseed plants have D12 and D15 desaturases which
act on phospholipid-bound OA. In the body, LA and ALA are further con-
verted to ARA and EPA, respectively and EPA is a precursor to DHA
(Sprecher 2000).
The eicosanoid family of metabolites which include prostaglandins,
leukotrienes and thromboxanes (Funk 2001; Smith 2005) are formed
directly from ARA and EPA. These molecules are key control points
for metabolic processes such as inflammatory responses, blood clot
induction and regulation of blood pressure (Yaqoob 2003). Inflammatory
response is regulated by the balance of these types of eicosanoids with
ARA- and EPA-derived eicosanoids producing a pro- and anti-inflam-
matory response, respectively (Calder 2003; Simopoulos 2006; Smith
2005). When EPA is not consumed directly in the diet, the ratio of LA
to ALA consumed has a direct affect on the concentration of ARA and
EPA because humans lack an o-3 desaturase that would convert LA to
ALA or ARA to EPA. Therefore, the essential need for LA and ALA in
the diet is in part due to their importance as intermediates of LCPUFAs
and eicosanoid biosynthesis, which are crucial for normal human
physiology.
Although inflammatory responses are critical in many human defense and
healing mechanisms, prolonged induction of an inflammatory state could lead
to health problems. In Western society, consumption of foods rich in LA, such
as vegetable oils or grain-fed meat and poultry, has increased substantially over
the past 60 years (Hibbeln et al. 2004). This increase combined with increased
consumption of hydrogenated fats where o-3 fatty acids have been selectively
removed, has resulted in a substantial shift in the balance of ARA and EPA in
the blood stream with numerous potential negative consequences (Hibbeln
et al. 2004; Simopoulos 1999; Wada et al. 2007) including those disease states
related to excessive inflammation or auto-immune conditions. For example,
inflammatory bowel disease has been linked to increased concentrations of
ARA (Ramakers et al. 2007).
Consumption of EPA and/or DHA has also been shown to be beneficial in
other areas of human health such as cardiovascular disease and mental health
(Ruxton et al. 2007). Benefits of EPA/DHA in decreasing death rates after a
myocardial infarction have been described while consumption of ALA is not as

effective (Breslow 2006; von Schacky and Harris 2007). EPA and DHA are
protective at doses <1 g/d due to suppression of fatal arrhythmias and at doses
>3 g/d, EPA plus DHA can improve cardiovascular disease risk factors,
including decreasing plasma triacylglycerides, blood pressure, platelet aggrega-
tion, and inflammation, while improving vascular reactivity (Breslow 2006; von
Schacky and Harris 2007). Low levels of EPA and/or DHA have been linked to
higher instances of depression and increased consumption of EPA and/or DHA
can attenuate symptoms of depression and bipolar disorder (Sontrop and
Campbell 2006). DHA is an important component of cell membrane phospho-
lipids (Horrocks and Farooqui 2004). Mammalian retinal and brain mem-
branes are enriched in DHA and it is important for the cognitive development
of infants (Fleith and Clandinin 2005; Iribarren et al. 2004; Stoll et al. 2001;
Willatts and Forsyth 2000). The use of DHA in treating brain-related diseases
such as Alzheimer’s disease have also been suggested (Oksman et al. 2006).
The best direct source of EPA and DHA in our diet comes from the
consumption of marine fish or their oils. In fact, the American Heart Associa-
tion has recommended at least two weekly servings of fish as part of a heart-
healthy diet. Many consumers are also complementing diet with fish oil supple-
ments. But, while increasing consumer awareness towards the health benefits of
fish oil, o-3 fatty acids, EPA and DHA is generally good from a health
perspective, the increasing demand for fish and fish oil products is placing
further stress on limiting fish populations, many of which are already in
jeopardy (Pauly et al. 2002). Additionally, many fish species and resulting fish
oil derived from them are tainted with contaminants and some consumption
restriction recommendations have been made (Mozaffarian and Rimm 2006).
In fact, the main source of LCPUFA in fish originates in marine microorgan-
isms such as diatoms, golden-brown algae, green algae, blue-green algae, micro-
bial fungi and dinoflagellates, all of which are rich in LCPUFA (Radwan 1991;
Shaw 1966). These organisms are ingested by small fish which in turn are eaten
by larger fish and other animals. Sustainable alternatives to fish oils where
LCPUFAs are produced through fermentation of microalgae are free from the
contaminants found in fish oils but their widespread use is limited to high value
applications such as infant formula and medical foods (Boswell et al. 1996) due
to their high cost of manufacture.
2.3.2 LCPUFA Production in Plants
In nature, there exist two main biochemical pathways to ARA, EPA and DHA.
These are the aerobic desaturase/elongase-type pathway and the anaerobic
polyketide synthase (PKS) pathway (Sperling et al. 2003; Bentley and Bennett
1999). The aerobic desaturase/elongase-type pathway can further be divided
into two classes based on whether the first step of the pathway is elongation or
desaturation.
2.3.3 EPA Production in Plants via the D6 Desaturase Pathway
In animals, including fish and humans, and the majority of marine micro-
organisms studied thus far, EPA is generated by a D6 desaturase pathway
(Sayanova and Napier 2004) where a double bond is first added to ALA by
a D6 desaturase to form stearidonic acid (STA), followed by elongation of
STA to eicosatetraenoic acid (ETA) catalyzed by a D6 fatty acid elongase
and lastly the formation of another double bond in ETA by a D5 desaturase
to form EPA. Some micro-organisms that produce ARA do so by an
analogous pathway as for EPA but the desaturases and elongases utilize
the o-6 forms of these substrates when they are present. Therefore, LA can
be converted to g-linolenic acid (GLA) by the D6 desaturases, GLA can be
converted to dihomo-g-linolenic acid (DGLA) by the elongases and DGLA
can be converted to ARA by the D5 desaturases. The substrate preference
for either o-6 or o-3 substrates varies depending on the species from which
the enzymes are derived and some species produce both o-6 and o-3 fatty
acids (Sayanova et al. 2006; Sayanova and Napier 2004). In addition, some
organisms can convert o-6 fatty acids (C18 and/or C20) to o-3 fatty acids
through the action of an o-3 fatty acid desaturase (Damude et al. 2006;
Oura and Kajiwara 2004; Pereira et al. 2004b; Sakuradani et al. 2005;
Spychalla et al. 1997). The ratio of products produced in any given pathway
will, therefore, be a function of both the substrate specificity of the enzymes
used and the concentrations of either o-3 or o-6 substrates available in the
host.
Most oilseed plants that produce polyunsaturated fatty acids also pro-
duce a very high concentration of LA, which results in a ratio of ARA to
EPA that is too high when converted in the body. One exception to this is
linseed oil, which has ALA concentrations of approximately 50–60% of
the total fatty acids. A transgenic approach to make higher levels of ALA
in soybean by expressing a bifunctional D12/D15 desaturase from Fusarium
moniliforme resulted in ALA concentrations as high as 72% in seed
(Damude et al. 2006). In a similar attempt to make pork healthier, trans-
genic pigs were produced, which expressed a ‘‘humanized’’ Caenorhabditis
elegans o-3 desaturase (Lai et al. 2006) and this resulted in pork fat with
higher concentrations of ALA.
The first step of the D6 desaturase pathway is the conversion of ALA to
stearidonic acid (STA). In humans, the D6 desaturase is rate-limiting (Burdge
et al. 2002; James et al. 2003). Thus, even when ALA concentrations in the diet
are high, ALA is poorly converted to EPA in healthy subjects (James et al. 2003;
Miles et al. 2004). Further, D6 activity has been shown to decline with age
(Bourre et al. 1990).
By-passing the ineffective D6 desaturase by consuming oils rich in STA
directly has been proposed as an indirect way of more effectively obtaining
needed levels of EPA and DHA. Some plants such as hemp, borage, black
currant and evening primrose express a D6 desaturase and produce GLA and
STA in their seed oils naturally, and these oils are currently marketed and sold
for their proposed health benefits (Barre 2001). But, their cultivation is carried
out on a scale that is relatively small and yields are poor making the oils
produced expensive.
Producing STA in commercial oilseed crops has also been proposed and
requires the minimal expression of a single D6 desaturase. The synthesis of GLA
and STA in a plant (tobacco) was first demonstrated using the D6 desaturase
from Synechocystis expressed constitutively under control of the 35S promoter
(Reddy and Thomas 1996). Subsequent expression of other D6 desaturases in
tobacco, Brassica juncea and soybean further improved yields of both GLA and
STA (Sayanova et al. 1997; Hong et al. 2002; Qiu et al. 2002; Sato et al. 2004).
STA production was further optimized in soybean using seed-specific expres-
sion of the borage D6 desaturase and the Arabidopsis D15 desaturase giving
STA contents of as high as 30% (Eckert et al. 2006). Both GLA and STA
production in oilseeds are under active commercial development with reports of
GLA synthesis in safflower yielding up to 73% GLA (Knauf et al. 2006) and
STA synthesis in soybean giving greater than 20% STA with about 5–6% GLA
(Wilkes 2007).
Producing GLA and STA in the seeds of commercial oilseed plants are
significant achievements in their own right, but STA still needs to be
consumed in larger amounts to have the same efficacy as EPA (Miles
et al. 2004). In addition STA does not appear to be converted by the
body to DHA, an n-3 LCPUFA that is very important for cognitive func-
tion (James et al. 2003). Thus, direct consumption of EPA and DHA in the
diet remains preferred and requires the expression of the remaining pathway
genes in plants.
In the D6 pathway to EPA, STA is elongated by two carbons to ETA via a
microsomal fatty acid elongation complex (Metz et al. 2001), which is similar to
that in the plastid but which uses acyl-CoA substrates instead of acyl-ACP
substrates. With the exception of a recently characterized elongase from the
marine parasitic protozoon Perkinus marinus, all of the elongases that are
involved in LCPUFA biosynthetic pathways are of the ELO/SUR4 gene family
(Venegas-Caleron et al. 2007). Of the remaining proteins involved in elonga-
tion, putative beta-ketoacyl-CoA reductases (Beaudoin et al. 2002; Han et al.
2002) and enoyl-CoA reductases (Gable et al. 2004; Paul et al. 2007) have been
suggested for yeast and plants. Recently, the PHS1 gene has been suggested to
be responsible for the dehydration reaction in yeast (Denic and Weissman
2007). Further desaturation of ETA by a D5 desaturase generates the final
EPA product.
Production of EPA in a plant using the D6 desaturase pathway was first
demonstrated in soybean (Kinney et al. 2004) and is, to date, the highest
abundance of EPA achieved in any plant tissue. In the study, a D6
desaturase, elongase and D5 desaturase gene from Mortierella alpina
were used along with an Arabidopsis fad3 gene (Yadav et al. 1993) and a
S. diclina D17 desaturase (Pereira et al. 2004b) and each were expressed
under the control of different, strong, seed-specific promoters. EPA con-
tents in embryos as high as 13% in embryos and 20% in seed were
achieved with little to no ARA produced due mainly to the presence of
the highly efficient D17 desaturase used. The o-3 to o-6 fatty acid ratio
increased from 0.2:1 (the normal soybean ratio) to 1.5:1 (a ratio close to
that of many fish oils) and overall elongation was 65% suggesting a highly
efficient transfer to CoA pools for subsequent elongation. Unexpectedly,
docoaspentaenoic acid (DPA), DHA precursor, was also found in the high
EPA lines as abundant as 4%. DPA resulted from the additional activity
of the M. alpina D6 elongase towards the D5 fatty acid EPA. This same
elongase had almost no D5 EPA-elongating activity when expressed in
yeast (Parker-Barnes et al. 2000). In the same study, some events where
the S. diclina D17 desaturase was not functioning contained ARA concen-
trations as high as 26% in seed were obtained (Damude unpublished data).
o-3 to o-6 ratios were further improved when the Arabidopsis o-3 desaturase
was replaced with a novel, bifunctional D12/D15 desaturase from Fusarium
moniliforme (Damude and Yadav 2005; Damude et al. 2006). The Fusarium
D15 desaturase had broad substrate specificity for numerous o-6 fatty acids
including LA>GLA>DGLA>ARA (Damude et al. 2006), and when co-
expressed with the LCPUA pathway, led to an overall o-3 content as high as
57% in soybean embryos with up to 16% EPA.
Interestingly, a recent similar attempt at producing ARA using the D6
desaturase pathway in soybean (Chen et al. 2006) resulted in low con-
centrations of ARA (2.1% of total lipids) in embryos and even lower
concentrations (0.5–0.8%) in seed. In this approach, a FAD3 down-
regulation cassette was combined with the Mortierella alpina D6 desatur-
ase, elongase and D5 desaturase genes, under control of the strong seed-
specific b-conglycinin promoter. Use of the same promoter multiple times
was suggested to cause poor expression of the genes and led to low ARA
concentrations.
The initial demonstration of EPA in soybeans using a D6 desaturase
pathway (Kinney et al. 2004) was followed closely by another report
(Abbadi et al. 2004) where ARA and EPA were produced (less than 2%
total) in tobacco and flax seeds using genes from the diatom Phaeodactylum
tricornutum and the moss Physcomitrella patens (Abbadi et al. 2004). In this
study, the EPA pathway intermediates GLA and STA predominated at
approximately 30% with only low concentrations of elongated fatty acids
such as DGLA, ETA, ARA and EPA. Results indicated the elongation step
was severely limited (10% total elongation) in both tobacco and flax. The
elongation bottleneck was shown to be the result of low incorporation of
the substrates for elongation, GLA and STA, into the acyl-CoA pools from
the phospholipid pools from where they were produced. This poor exchange
resulted in the incorporation of GLA directly into triglyceride mostly
through the direct conversion of phosphatidylcholine (PC) containing
GLA to DAG followed by acylation to TAG. A similar direct incorporation

of phospholipid backbones containing the unusual fatty acid eleostearic acid
directly into TAG was seen when a Momordica charantia fatty acid
conjugase was expressed in Arabidopsis (Cahoon et al. 2006).
In another study, a full complement of D6 desaturase pathway genes has
been expressed in Brassicca juncea (Wu et al. 2005) and inclusion of a D17
desaturase significantly increased EPA production (up to 15%) with a substan-
tial decrease in ARA. In all of these studies described above, the D6 and D5
desaturases included utilized acyl-phosholipid substrates and in some cases
transfer of the acyl groups to acyl-CoA pools was shown to be limiting. One
other D6 desaturase pathway study in Arabidopsis seed used a D6/D5 desaturase
from a species of zebrafish (Danio rerio) which utilizes acyl-CoA substrates
along with a Caenorhabditis elegans elongase (Robert et al. 2005). It was
thought that a complete pathway with preference for acyl-CoA substrates
would improve flux since reduced exchange between acyl-CoA and phospholi-
pid pools would be required. Using this approach, an EPA content of only 2.5%
was achieved.
2.3.4 EPA Production in Plants via the D9 Elongase Pathway

Species from the Prymnesiophyceae (e.g. Pavlova, Isochrysis) Acanthamoebidae
(e g. Acanthamoeba) and Euglenophyceae (e.g. Euglena) families of LCPUFA-
producing microorganisms, have been shown to use a slightly different aerobic
pathway to produce EPA (Sayanova and Napier 2004). In this alternate D9
elongase pathway, ALA is first elongated by a D9-specific elongase to eicosa-
trienoic acid (ERA), followed by D8 desaturation to ETA. As in the more
common D6 pathway, these fatty acids are then desaturated to EPA respectively
by a D5 desaturase.
The earliest published report of synthesis of EPA in plants described
expression of this alternate D9 elongase pathway in Arabidopsis leaf
tissue. A D9 elongase from the microalgae Isochrysis galbana, a D8
desatuarase from the Euglenophyceae, Euglena gracilis, and a D5 desatur-
ase from the microbial fungus Mortierella alpina were expressed consti-
tutively in the model plant Arabidopsis (Qi et al. 2004). EPA contents as
high as 3.0% with ARA contents up to 6.6% were achieved in Arabidop-
sis leaves with ratios of o-3 to o-6 ratio fatty acids (2.2:1) similar to that
commonly found in fish oils (Sargent 1997). Total elongation of fatty
acids was good at 36% demonstrating a relatively high availability of LA
and ALA in the acyl-CoA pool of Arabidopsis leaves. It was suggested
that the bottleneck of acyl-transfer between acyl-CoA and phospholipid
pools was relieved by placing the acyl-CoA-requiring elongation step first
in the pathway followed by two acyl-phospholipid-requiring desaturation
steps.
In a case of premature desaturation, the fatty acid by-products sciadonic

acid (SCI) and juniperonic acid (JUP) were also formed in this D9 elongase
pathway. This resulted from the direct action of the D5 desaturase on EDA and
ERA before the D8 desaturation step. Interestingly, the species from which the
D9 elongase and D8 desaturase were taken do not produce significant levels of
these fatty acid by-products suggesting different specificities of the host D5
desaturase or more efficient flux through the pathway.
Some plant gymnosperm species (e.g. Pinaceae family) naturally produce
seeds with twenty carbon fatty acids having D5 double bonds such as sciadonic
acid (SCI) and juniperonic acid (JUP), suggesting the presence of C20 elongases
and D5 desaturases (Wolff et al. 2000).
2.3.5 DHA Production in Plants via the Aerobic Elongation/

Desaturation Pathways
In animals, generation of DHA from EPA occurs by the Sprecher pathway

whereby EPA is elongated twice by the fatty elongation complex (Sprecher
et al. 1999) and the first enzyme of elongation is specific for the co-acylated
form of EPA. The 24 carbon fatty acid tetracosapentaenoic acid (TPA) generated
by elongation is then desaturated by the rate-limiting D6 desaturase to produce
tetracosahexaenoic acid (THA). Chain reduction by 2 carbons via beta-oxidation
forms DHA (Wallis et al. 2002).
In microorganisms, DHA is synthesized by a much simpler pathway
from EPA where initial elongation to docosapentaenoic acid (DPA) by the
fatty acid elongation complex is followed by a D4 desaturase to directly
produce DHA without the need for further elongation, D6 desaturation or
beta-oxidation (Sayanova and Napier 2004).
The first successful production of DHA in plants was carried out in
soybean (Kinney et al. 2004). The D5 elongase from Pavlova sp. (Pereira et
al. 2004a) and the D4 desaturase from Schizochytrium aggregatum (Mukerji
et al. 2002) was added alongside the D6 desaturase EPA biosynthetic
pathway, and soybean somatic embryo oil with up to 3.3% DHA was
achieved. To this date this continues to be the highest concentration of
DHA made in any plant. It was difficult to separate whether the additional
Pavlova elongase or the dual specificity of the M. alpina D6 elongase for
D5 substrates contributed to the elongation of EPA but given the high
levels of DPA achieved with only the M. alpina elongase, it is most likely
the latter. The S. aggregatum D4 desaturase used was highly active in
plants with, in some cases, close to 100% conversion of DPA to DHA.
Subsequently, DHA has been produced in other plants including up to
1.5% in Brassica seed by combining EPA biosynthetic genes (D6 desaturase
pathway) with the D4 desaturase from Thraustochytrium sp., and an elon-
gase from the fish Oncorhynchus mykiss (Wu et al. 2005) and up to 0.5% in
Arabidopsis seeds by re-transforming an Arabidopsis plant making EPA with

a D5 elongase and a D4 desaturase from Pavlova salina (Robert et al. 2005).
2.3.6 DHA Production in Plants via the Anaerobic Polyketide

Synthase Pathway
Many marine microbes synthesize EPA or DHA using the anaerobic polyketide
synthase pathway (Metz et al. 2001), which are similar to the fatty acid synthase
complex in plants. These enzymes can be formed from multiple large proteins
which contain many multi-domained subunits and where each domain carries
out a different chemistry. Double bonds are produced in the growing fatty acid
chain by a dehydrase-isomerase mechanism similar to FabA in E. coli and do
not require oxygen as do fatty acid desaturases (Metz et al. 2001). Interestingly,
in some organisms both a PKS and a fatty acid synthase pathway for EPA or
DHA synthesis may be present. For example, a complete PKS-type DHA-
synthase has been cloned and characterized from a number of Thraustochytid
species, as have various fatty acid desaturases and elongases (Metz et al. 2004;
Qiu et al. 2001).
Successful expression of PKS genes in yeast and plants has been reported
(Metz et al. 2006, 2007). Genes encoding the three subunits (ORFA, B, C) of a
Schizochytrium PKS were co-expressed with a phosphopantetheinyl transfer-
ase (PPT) from Nostoc, essential for activating the ACP domains of the DHA-
synthase PKS individually under control of the linin seed-specific promoter
from flax. Expression in the plastid was achieved by fusion with a Brassica
acyl-ACP thioesterase plastid targeting signal. Arabidopsis seeds were
obtained having up to 0.8% DHA with an additional 1.7% DPAn-6, which
is also observed in Schizochytrium. Further increases in DHA content in
Arabidopsis seed were made by co-expressing a PKS with either an acyl-CoA
synthetase (ACSI or ACSII) from Schizochytrium, or RNAi constructs for
down-regulation of the Arabidopsis KASII or KASIII genes. Analysis of the
fatty acid profiles of single seed expressing the PKS, PPT and the KASIII
RNAi construct showed levels of DHA and DPAn-6 as high as 2.4 and 1.8%,
respectively.
2.4 Modifying Vegetable Oils for Non-food Purposes
2.4.1 Non-food Uses of Plant Oils
Consumption of soybean oil for industrial uses has undergone a dramatic

increase between 2003 and 2006. During this period, the use of soybean oil for
industrial applications increased from 552 million pounds to 2379 million
pounds in the United States (SoyStatsTM 2007). Currently, 12% of U.S.
soybean oil consumption is directed to industrial uses. The large majority of this
increase has resulted from demand for soybean oil for biodiesel production.
Given the rising petroleum prices, it is likely that this demand will increase into
the foreseeable future, not only for biodiesel but for the production of industrial
materials such as lubricants, paints, and plastics that have historically been
derived from crude oil.
2.4.2 High Oleic Acid Soybean Oil
Nearly all of the soybean oil that is now used for industrial applications is
conventional soybean oil that lacks any genetic modification of its fatty
acid composition. However, through the use of breeding and biotechno-
logy, it is possible to generate fatty acid profiles that improve the func-
tionality of soybean oil for industrial uses, including biodiesel. Soybeans
with increased oleic acid content have received the most attention for use
in a variety of industrial applications (Kinney and Clemente 2005).
Enhancement of oleic acid content can be achieved by breeding different
mutant alleles for D12 oleic acid desaturase (FAD2) genes (Burton et al.
2004). Typical FAD2 enzymes convert oleic acid to linoleic acid (Okuley
et al. 1994). This enzyme uses an oleic acid molecule principally to phos-
phatidylcholine (PC) as its substrate. By mutating or suppressing the
expression of FAD2 genes, soybean seeds accumulate oleic acid, rather
than linoleic acid, as the major component of the seed oil. The largest
increases in oleic acid have been obtained through a biotechnological
approach by suppression of the FAD2-1 gene in combination with down
regulation of palmitoyl-acyl carrier protein thioesterase (FatB) genes (Buhr
et al. 2002). Through the use of this approach, high oleic (HO) acid oils
containing as much as 90% oleic acid have been achieved. The suppression
of FatB expression also reduced the palmitic acid content of these oils to
<4% of the total fatty acids (Buhr et al. 2002), resulting in oils with less
saturated fatty acids.
HO soybean oils are not only enriched in oleic acid but also have reduced
amounts of the polyunsaturated fatty acids linoleic and linolenic acids. For
example, HO oils with as much as 90% oleic acid have <4% each of polyunsa-
turated fatty acids and palmitic acid (Buhr et al. 2002). It is likely that most of
the remaining polyunsaturated fatty acids can be removed by breeding into 1%
linolenic acid soybean mutant lines (Ross et al. 2000). The combination of high
oleic acid and low polyunsaturated fatty acid content results in a liquid oil with
greatly improved oxidative stability. HO soybean oil with 85% oleic acid, for
example, has an oxidative stability index value that is nearly 12-fold higher than
that of conventional soybean oil (Knowlton 1999). This property is critical for
the use of vegetable oils in lubricants, including motor and hydraulic oils (Erhan
et al. 2006; Sharma et al. 2005). HO soybean oil also displays superior
properties in biodiesel formulations compared to conventional soybean oil

(Kinney and Clemente 2005; Tat et al. 2007). The reduced content of polyunsa-
turated fatty acids in HO soybean oil results not only in enhanced oxidative
stability but also less emission of nitrogen oxides (NOx) (Tat et al. 2007). In
addition, the cold point properties are also improved in HO soybean oil due
primarily to its lower content of palmitic acid (4% in HO soybean oil versus
12% in conventional soybean oil) (Tat et al. 2007). For example, methyl esters
generated from HO soybean oil have cloud and pour points of –5 and –98C,
respectively. By comparison, the cloud and pour points of methyl esters produced
from conventional soybean oil are 1 and 08C, respectively (Tat et al. 2007).
Undoubtedly, soybean oils with additional novel industrial functionalities can
be generated by crossing of HO soybeans derived from biotechnology with
soybeans with altered contents of other fatty acids (e.g. high stearic acid) derived
from breeding of mutant alleles.
2.4.3 Metabolic Engineering of Soybean for the Production

of Oils with High-Value Industrial Fatty Acids
The improved properties of HO soybean oils for industrial applications demon-
strate the utility of altering the relative proportions of the five fatty acids
typically found in soybean seeds. Another approach for generating soybean
oil with improved industrial functionality is to introduce genes that produce
fatty acids with novel structures. Such genes have typically been isolated from
non-agronomic species that accumulate unusual types of fatty acids in their
seed oils (Voelker and Kinney 2001). The enzymes encoded by such genes have
been useful for producing fatty acids with carbon chain modifications such as
hydroxy or epoxy groups or for producing fatty acids with novel double bond
positions and configurations or for even producing fatty acids with chain
lengths greater than 18 carbon atoms. Many of these genes encode enzymes
that are related to FAD2 but have evolved new enzymatic properties
(Cahoon and Kinney 2005). One example is the oleic acid hydroxylase from
castor (Ricinus communis) (van de Loo et al. 1995). This enzyme uses the same
oleoyl-phosphatidylcholine substrate as the typical FAD2 but introduces a
hydroxyl group rather than a cis double bond at the D12 position. The hydro-
xylated fatty acid product ricinoleic acid (OH-18:1D9) has a wide-range of
useful industrial properties. These include uses in lubricants, hydraulic fluids,
surfactants, cosmetics, and nylon production. The hydroxyl group of ricinoleic
acid may also increase the lubricity of fatty acid methyl esters in biodiesel
applications (Kinney and Clemente 2005). In an attempt to capture these
desirable industrial properties in soybean, research has been conducted to
introduce the castor hydroxylase gene under control of a seed-specific promoter
to soybean (Kinney and Clemente 2005). These studies have resulted in the
generation of soybeans that accumulate ricinoleic acid to approximately 15% of
the total fatty acids of the seed oil. Oils extracted from these engineered seeds
are currently being evaluated to determine the value of the increased lubricity
associated with fatty acid hydroxylation and the improved oxidative stability
associated with the increased oleic acid content (Clemente unpublished results).
Fatty acid epoxidation is another modification that has received interest for
the improvement of the industrial value of soybean oil. Chemical epoxidation of
soybean oil is currently used to generate plasticizers and precursors such as
polyols for the production of coatings, adhesives, and biopolymers (Liu et al.
2006). Epoxidation involves the reaction of the double bonds of the fatty acids
of soybean oil with hydrogen peroxide under acidic conditions (Vlcek and
Petrovic 2006). This reaction is non-specific for conversion of the D9, 12, and
15 double bonds that can be found in the fatty acids of soybean oil. A number of
non-agronomic plant species have evolved enzymes that allow for the specific
conversion of the D12 double bond of linoleic acid to form the epoxy fatty acid
vernolic acid (Voelker and Kinney 2001). Vegetable oils enriched in vernolic
acid have received interest not only for existing applications for of epoxidized
soybean oil but also for paint solvents with low content of volatile organic
compounds (VOCs) solvent for paint (Bhardwaj et al. 2007). Novel chemistries
for conversion of vernolic acid to industrially useful materials have also been
explored (Ayorinde et al. 1997). The D12 epoxy group of vernolic acid can be
produced from linoleoyl-PC by the activity of a divergent FAD2 epoxygenase
(Lee et al. 1998) or from a structurally unrelated cytochrome P450 epoxygenase
(Cahoon et al. 2002). A gene from Vernonia galamensis for a FAD2 epoxygen-
ase has been introduced into soybean to produce oils containing approximately
7% vernolic acid (Hitz 1998). Similar levels of vernolic acid have been produced
in soybean somatic embryos by expression of a cytochrome P450 epoxygenase
from Euphorbia lagascae (Cahoon et al. 2002).
A major industrial use of soybean oil is as a component of soy ink
(Erhan and Bagby 1991). Soy ink is widely used for the color print of news-
papers. A limitation of soybean oil for newspaper print ink is its relatively slow
drying rate. To improve its drying properties, soybean oil can be supplemented
with tung oil, which contains high levels of fatty acids with conjugated double
bonds (or ‘‘conjugated fatty acids’’). The double bonds of conjugated fatty acids
are positioned at adjacent carbon atoms whereas the double bonds of linoleic
and linolenic acids, the major polyunsaturated unsaturated fatty acids of soy-
bean oil, are separated by methylene groups. To date, soybeans have been
engineered to produce two isomers of conjugated fatty acids eleostearic acid
and calendic acid (Cahoon et al. 1999, 2006). Both fatty acids are produced by
the activity of FAD2-related enzymes termed ‘‘fatty acid conjugases’’
(Cahoon et al. 1999, 2001; Qiu et al. 2001). These enzymes convert an existing
cis-double bond of linoleic acid bound to PC into two conjugated trans-double
bonds to generate a conjugated trienoic fatty acid (Cahoon and Kinney 2005).
The fatty acid conjugase that produces calendic acid converts the D9 double
bond of linoleic acid to D8-trans and D10-trans double bonds; whereas, the fatty
acid conjugase that generates eleostearic acid converts the D12 double bond of
linoleic acid into D11-trans and D13-trans double bonds (Cahoon and Kinney
1995). A D9-modifying conjugase cDNA from Calendula officinalis has been
engineered in soybean to produce calendic acid at levels of 10–25% of the fatty
acids of the seed oil (Cahoon et al. 2006). Similarly, D12-modifying conjugase
cDNAs from Momordica charantia, Impatiens balsamina, and Chrysobalanus
icaco have been introduced into soybean somatic embryos to generate oils with
up to 20% eleostearic acid (Cahoon et al. 1999, 2006, 2007a).
From a technical standpoint, the metabolic engineering of genes from
other species has successfully resulted in the production of soybean oil
with novel fatty acid compositions. However, in most all cases to date, the
amounts of these fatty acids obtained in soybean oil have been consider-
ably lower than those in oils from seeds that naturally accumulate unusual
fatty acids. For example, castor bean accumulates ricinoleic acid to levels
of approximately 90% of its seed oil. However, soybeans engineered to
express the castor bean hydroxylase accumulate ricinoleic acid to amounts
of about 15% of the seed oil (Kinney and Clemente 2005). The inability to
achieve high levels of unusual fatty acid accumulation in engineered soy-
bean seeds has been the major technical hurdle that has limited the adop-
tion of this technology for producing new types of industrial oils in
soybean. In the case of fatty acids derived from divergent FAD2s, bottle-
necks associated with unusual fatty acid accumulation in engineered oil-
seeds appears to result from defects in the efficient flux of unusual fatty
acids from PC following their synthesis on this lipid (Cahoon et al. 2006,
2007b). This is exemplified by phenotypes observed in seeds engineered to
express fatty acid conjugases. In Arabidopsis and soybean seeds that
express D9- and D12-type conjugases, conjugated fatty acids not only
accumulate in TAG but are also present in PC at the same or higher
relative amounts (Cahoon et al. 2006). For example, soybean seeds that
express the C. officinalis conjugase accumulate calendic acid to amounts of
about 20% of total fatty acids in triacylglycerols (Cahoon et al. 2006).
Calendic acid also aberrantly accumulates to levels of about 25% of the
fatty acids in PC in these seeds. By contrast, calendic acid is found in
C. officinalis seeds at levels of approximately 55% of TAG fatty acids, but
<1% of the fatty acids of PC. As such, it appears that C. officinalis has
evolved a mechanism for limiting calendic acid aberrantly accumulation in
PC that is absent in soybean seeds. Seeds of other species that naturally
produce conjugated fatty acids have also apparently evolved the metabolic
capacity to efficiently remove these fatty acids from PC, as conjugated
fatty acids are rarely found at levels of >2% in PC, even in species that
accumulate conjugated fatty acids to >80% of the fatty acids of their seed
oil (Cahoon et al. 2006). Current research is focusing on the identification
of divergent types of phospholipolytic enzymes from plants that naturally
accumulate unusual fatty acids from the activity of FAD2-related enzymes.
It is presumed that such enzymes have evolved for the efficient metabolism
of unusual fatty acids, and that co-expression of these metabolic enzymes
along with FAD2 biosynthetic enzymes will enable the production of

economically-relevant levels of industrial fatty acids in soybean seeds.
Acknowledgments Research on unusual fatty acid metabolism in the Cahoon lab is supported
by the National Science Foundation (DBI- 0701919).
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Chapter 3
Soybean
Elroy R. Cober, Silvia R. Cianzio, Vincent R. Pantalone, and Istvan Rajcan
3.1 Introduction
Soybean [Glycine max (L.) Merr.] is the leading oilseed crop produced in the
world (Wilcox 2004). Over the past three decades, world production of soybean
has almost tripled, from 73,854,802 Mg in 1977 to 216,144,262 Mg in 2007
(FAOSTAT 2008). The majority of soybean is grown in North and South
America, China and to a smaller extent in many other countries on every
continent. During the past five decades, the USA has been the world’s leading
producer of soybean representing 33% of the world production, followed by
Brazil with 27%, Argentina with 21%, China with 7.2%, India with 4.4%,
Paraguay with 1.8% and Canada with 1.3% (FAOSTAT 2008). During the
1990s and 2000s production in Brazil and Argentina has seen a tremendous
increase, reaching almost 50% of the total world production when combined in
2007. Increasing world population, constant need for animal feed and over 300
different soybean products contribute to the strong demand for soybean in
world markets. From 1992 to 2007, soyfood sales increased from $300 million
to nearly $4 billion (Soyatech Inc. 2008) with most of the recent increase coming
from soymilk. In 2008, 85% of the consumers in the USA rated soybean as
healthy, which was up 3% from 2006 (United Soybean Board 2008). Healthy
aspects of soyfoods go beyond the oil and protein and include minor com-
pounds with nutraceutical properties such as isoflavones, saponins and toco-
pherols (Rajcan et al. 2005). Clearly, soybean production, consumer acceptance
and consumption in non-traditional regions of the world are on the rise.
Because of the high concentration of protein (40%) and oil (20%) typically
found in the seed, soybean is used both for oil production and protein proces-
sing. Most of the soybean is used for oil extraction and for animal feed
processed from the remaining meal after the oil is crushed. Additionally, a
significant and increasing portion is also used as food, including traditional
E.R. Cober (*)

Agriculture and Agri-food Canada, Eastern Cereal and Oilseed Crop Research Centre,
Ottawa, ON, Canada
e-mail: elroy.cober@agr.gc.ca

58 E.R. Cober et al.
uses originating in Asia such as tofu, miso, edamame, soy sauce, soymilk, natto
and newer uses in Western culture such as soy pudding, soy granola bars, soy
nuts and other products.
Typically, soybean oil consists of approximately 10% palmitic (16:0), 4%
stearic (18:0), 22% oleic (18:1), 54% linoleic (18:2) and 10% linolenic (18:3) acid
(Wilson 2004), making it one of the healthiest vegetable oils. In addition to oil,
the protein contains all essential amino acids and, therefore, is a prime source
for both animals and humans. However, soymeal remains deficient in the sulfur
containing amino acids, methionine and cysteine, and breeders are currently
striving to enhance their concentrations (Panthee et al. 2006).
Major improvements in soybean seed yield occurred during the past several
decades. In the USA soybean yields have risen by 22.6 kg ha–1 yr1 from 1924 to
1997, however, during the 25 years from 1972 to 1997, the increase has been
faster, 31.4 kg ha1 yr1 (Specht et al. 1999). During the same period in Ontario,
Canada, changes in some physiological traits have contributed to a 0.5% yield
increase yr 1, i.e. improvements in lodging resistance, N2 fixation, and stress
tolerance among other traits (Specht et al. 1999; Voldeng et al. 1997).
Breeding has played a dominant role in yield improvements coupled with
production practices such as increased planting density, which resulted from new
cultivars possessing smaller leaf area index and higher photosynthetic rates (Cober
et al. 2005), particularly in short-season soybean. The objective of this chapter is to
discuss a wide range of topics from the origin of the species and historical perspec-
tives up to current as well as future breeding goals and the use of new biotechno-
logical tools in soybean improvement. Unfortunately, it is not possible to cover
every trait under improvement. For a detailed review, the reader is referred to the
most recently published soybean monograph (Boerma and Specht 2004).
3.2 Origin and Domestication
The domesticated soybean [Glycine max (L.) Merrill] is a member of the

Fabaceae family, subfamily Papilionoideae, tribe Phaseoleae (Hymowitz
2004) (Fig. 3.1). Phaseoleae is the most important tribe for species used as
Family Fabacae
Subfamily Papilionoideae
Tribe Phaseoleae
Genus Glycine
Subgenus Soja
Glycine max (2n = 40)
A true domesticated species

Fig. 3.1 Taxonomic
classification of Glycine max
3 Soybean 59
food and feed sources, such as soybean, pigeon pea [Cajanus cajan (L.) Merr.],
and common bean, lima bean and tepary bean (Phaseolus spp.) among many
others.
The genus Glycine is composed of two subgenera, Glycine (perennials) and
Soja (annuals). The subgenus Soja includes the cultivated soybean, G. max, and
G. soja, the wild annual soybean relative. G. max is a true domesticate, the
species would not exist in the absence of human intervention.
Soybean originated in China, domestication of soybean took place 1500–1100
BC, or perhaps earlier (Palmer and Hymowitz 2004). It was probably grown in the
Korean peninsula as well as central China by the first century. From that time up to
the 15th to the 16th century, soybeans were introduced in Southeast Asia, and from
there to Europe before 1713. Soybean was introduced to North America in 1765.
Goldblatt (1981) indicated that ‘the basic chromosome number for
Phaseoleae is almost certainly x ¼ 11, probably basic in all tribes, empha-
sizing that aneuploid reduction to x ¼ 10 is prevalent through the Papilio-
noideae’. Hymowitz (2004) and his collaborators hypothesized that a
putative ancestor of the genus Glycine with 2n ¼ 20 arose in Southeast
Asia. This progenitor, however, is either extinct or has yet to be collected
and identified. Tetraploidization through auto- or allopolyploidy of the
progenitor species occurred either prior or after dissemination from the
ancestral region. Singh et al. (2001) proposed a more complete path of
migration from the ancestral region to China (Figs. 3.2 and 3.3). The
common progenitor is assumed to be a wild perennial (2n ¼ 4x ¼ 40,
unknown or extinct) that later evolved to a wild annual (2n ¼ 4x ¼ 40,
G. soja) and finally to the domesticated soybean (2n ¼ 4x ¼ 40, G. max,
cultigens). All described species of the genus Glycine exhibit normal diploid
meiosis, and are primarily inbreeders (Singh and Hymowitz 1985).
Cultigen annual (2n = 4x = 40) Glycine max
Wild annual (2n = 4x = 40) Glycine soja
Wild perennial (extinct?)

(2n = 4x = 40)
Fig. 3.2 Ancestors of the

cultivated soybean
Wild ancestor
Glycine soja
Unknown
ancestor
Domesticated soybean
Glycine max
Fig. 3.3 A pictorial of the ancestry of the domesticated soybean and the profound changes in
plant phenlogy and morphology by domestication
Genomic relationships among diploid species of the subgenus Glycine have

been extensively studied and established by cytogenetic analyses, biochemical
techniques and molecular methods. Different genomes have been differentiated
among several of the Glycine spp., and identified as AA, A1A1, BB, B1B1, and
B2B2, depending on the species. For a scholarly review of these and related
topics the reader is referred to the chapter by Hymowitz (2004).
In the cultivated soybean, the gene pool concept developed by Harlan and de
Wet (1971), identifies a primary gene pool constituted by soybean cultivars,
landraces and Glycine soja. Hybridization among them creates vigorous plants,
with normal meiosis and complete seed fertility. Gene segregation is also
normal. The secondary gene pool is formed of yet unknown species, and to
date the cultivated soybean does not possess one. The tertiary gene pool is
formed by the wild perennial species. Production of hybrids between members
of the primary gene pool and those of the tertiary gene pool is difficult, where
survival of the F1 seed requires use of special embryo rescue techniques.
3 Soybean 61
In spite of its economic importance, the basic study of the soybean species
has lagged behind many others (Hymowitz 2004). Soybean chromosomes are
smaller than chromosomes of most crop plants, making difficult the conduct of
basic studies. Additionally, it is not considered a model plant for cytogenetic
studies because its chromosomes are high in number (2n ¼ 40), small in size, and
morphological similar lacking distinguishing landmarks. Molecular research
has established 20 linkage groups which are not yet associated with their
respective chromosomes (Singh et al. 2007). However, newly developed stocks
have been formed and it is expected that these, together with new technologies,
will provide the tools to conduct genetic studies that will benefit soybean
breeding, production and physiology research.
3.3 Varietal Groups
As mentioned earlier, it is believed that the cultivated soybean [Glycine max (L.)
Merr.] originated in China by domestication of the wild progenitor species,
Glycine soja. In order to understand the varietal groups of soybean, it is
important to look at the genetic diversity within different gene pools worldwide.
Evaluation of genetic diversity has traditionally been conducted using differ-
ences in morphological and agronomic traits and/or pedigree (Nelson et al.
1987; Sneller 1994; Gizlice et al. 1994). A limitation of evaluating genetic
distances based on agronomic traits is their often high dependence on the
testing environment, which greatly reduces the range of soybean genotypes
that could be compared directly (Li and Nelson 2001). To overcome this
issue, a core set of randomly amplified DNA polymorphism (RAPD) markers
was developed to study a broad spectrum of genetic diversity among soybean
accessions (Thompson and Nelson 1998).
Perhaps not surprisingly, studies have shown that genetic distance within the
progenitor G. soja was much greater than within G. max but smaller than between
groups of G. soja and G. max (Li and Nelson 2002). The authors also observed
that much greater variation was found within relatively small geographic regions
in China in G. soja accessions than for G. max from the same province (Li and
Nelson 2002). Molecular genetic analysis of U.S. and Chinese soybean ancestral
lines indicated that cluster analysis clearly separated the ancestral gene pools of
China and USA (Li et al. 2001). Clusters reflected the geographic origin of the
lines tested and showed that large differences existed between northern U.S. and
Chinese ancestral lines on the one hand, and between the central and southern
Chinese ancestral lines on the other (Li et al. 2001).
Within Chinese germplasm, the coefficient of parentage was used to deter-
mine genetic diversity in soybean cultivars released from 1923 to 1995 (Cui et al.
2000). Cultivar pools from three different growing regions in China (Northeast,
Northern and Southern China) were completely unrelated and were also influ-
enced by crossing systems and release eras (Cui et al. 2000). Another research
group reported that the mean genetic distance in soybean cultivars within China
was much larger than the distance within Japan or South Korea but smaller
than that between China and S. Korea or Japan (Li and Nelson 2001).
Attempts have been made to use the diversity of varietal groups from
different regions and/or alleles from G. soja to improve germplasm diversity
and seed yield in the cultivated soybean. Yield quantitative trait loci (QTL)
were identified in crosses involving an exotic and adapted parent (Mansur et al.
1996; Orf et al. 1999a; Orf et al. 1999b; Smalley et al. 2005). In 2004, Kabelka
et al. reported 15 QTL significantly associated with yield in a cross between
‘BSR 101’, which has nine of the 10 ancestral lines contributing to North
American gene pool, and the experimental line LG82-8379, developed from a
cross between two plant introductions (PIs). For nine of the QTL, the exotic
parent contributed the high yielding allele (Kabelka et al. 2004) suggesting the
genetic potential that exotic germplasm sources may have to further improve
seed yield. Using three backcross derived populations, another group reported
13 yield QTL, of which 8 carried a high yielding allele from PI, that mapped to
previously reported yield QTL (Guzman et al. 2007). The same group also
found significant QTL x environment interaction, probably due to undetectable
or weak QTL effects in some environments (Guzman et al. 2007). It is expected
that further attempts by different research groups world-wide will result in the
finding of more yield QTL coming from exotic sources and further enhance-
ment of genetic diversity in soybean breeding gene pools for yield and other
traits’ improvement.
Soybean is an autogamous species and all former and current cultivars are
inbred lines. However, heterosis has been reported in soybean. A summary of
14 experiments conducted since 1930, reported average mid-parent heterosis
(MPH) for yield grain ranging from þ 14 to þ 46%, whereas high-parent
heterosis (HPH) ranged from þ 4 to þ 34% (Palmer et al. 2001). Despite the
existence of genetic male sterility and heterosis expression in soybean, no
soybean hybrids are used in commercial production. In China, Prof. Sun
Huan from the Jilin Academy of Agricultural Sciences recently released hybrids
‘HybSoy 1’ and ‘HybSoy 2’, grown in 200 ha demonstration plots, however, the
continuing pollination efficiency problems for seed production have hampered
their full release (Sun Huan, Jilin Academy of Agricultural Science, China; pers.
comm.). Unless better or more efficient pollinator systems can be found, the
genetic male sterility used to develop hybrids will not suffice for the commercial
release of hybrid soybean.
3.4 Genetic Resources
Success of the crop is credited to thousands of growers from antiquity to today,

and to countless number of researchers in many disciplines who have contrib-
uted with plant selections, knowledge and work to improve the species. Soybean
3 Soybean 63
was domesticated from the wild soybean, which is an annual weedy-form

climber (Fig. 3.3), whose pods contain black seeds that shatter at maturity
(Singh et al. 2007).
An estimated 45,000 unique Asian landraces have been collected and are
maintained in G. max collections around the world (Carter et al. 2004). These
large collections are becoming actively searched, studied and characterized, as
new challenges related to plant health and seed production acquire economic
importance. The studies will determine the value of the genetic diversity and
possible future use.
China possesses the largest collection of G. max accessions, 23,578, followed
by the US with slightly over 18,000, and the Asian Vegetable Research and
Development Center (AVRDC) with 12,508 accessions (Carter et al. 2004).
Japan, Russia, Ukraine, India and Brazil, also hold sizable collections,
although the actual number of accessions in each is smaller. Many studies on
genetic diversity and on the importance of collection and preservation of
soybean land races have been done and still are in process. And as new
technologies are developed, there is no doubt that even old concerns will be
revisited. In the interest of space, however, only some information will be
discussed in this section, as an attempt to open up interest areas to the readers.
For an extensive review of the subject up to 2004, the reader is referred to the
publication by Carter et al. (2004).
In China where the center of origin of the species is located, the collections
are actively characterized. Dong et al. (2004) used the database of the National
Germplasm Evaluation Program of China (NGEPC) to study geographical
distribution of accessions, genetic diversity of 15 qualitative and quantitative
traits, and genetic diversity of centers of the cultivated soybeans. Of the 22,637
known accessions studied 20,570 had been collected between latitudes 188 and
538 N and longitudes 808 and 1368 E. The authors reported that broader genetic
diversity was found in accessions collected between 348 to 418 N and 1108 to
1158 E, which prompted them to propose one genetic diversity center located
downstream of the Yellow River Valley. Northeast China is also thought to be
one of the regions in which soybean originated, particularly due to the existence
of a number of valuable soybean varieties in the region (Yamanaka et al. 2004).
To determine genetic diversity in this region, Yamanaka et al. (2004) studied a
group of 3,000 accessions measuring an array of quantitative and qualitative
traits, along with DNA determinations. Large variations were observed in traits
such as plant height (7–227 cm), protein (11.5–59.4%), and oil contents
(10.3–23.6%). These results indicate the potential value of genotypes in the
collection for use as breeding materials. The authors also used SSR markers in
253 accessions to determine genetic diversity at the molecular level. Of the 253
accessions, 194 varieties were from Northeast China, and 59 from Japan. SSR
markers indicated the two groups are genetically distantly related, and that
Chinese accessions are clearly rich in diversity at the DNA level. A study
conducted by Chen and Nelson (2005) using RAPD markers, identified six
major clusters of varieties that were genetically different although varieties
within each cluster were similar probably due to the fact they were obtained
from the same geographical location. Results provided evidence that primitive
cultivars of soybeans in China were genetically isolated in relatively small
geographical areas. Similar results have also been reported by other researchers
(Carter et al. 2004).
Estimates of genetic diversity in the annual wild soybean collection in China
have also been obtained (Dong et al. 2001; Ru et al. 2006). Both studies
identified genetic diversity in the G. soja collections using various diversity
indices, applied to morphological and chemical traits (Dong et al. 2001) and
to molecular markers, such as AFLP, ISSR, and SSR (Ru et al. 2006). The
results obtained by Dong et al. (2001) also suggested the possibility of three
centers of origin for G. soja in China: the Northeast, the Yellow River Valley,
and the Southeast Coast of China. On the basis of the genetic diversity esti-
mates, geographical isolation was and still is an effective mean to conserve
diversity. The research groups coincide on the importance of conserving genetic
diversity within the cultivated and wild annual soybean collections. These
observations reinforce the potential use of the collections for gene-mining,
and the need to preserve genetic information and genetic types.
In the US, the search for genetic diversity has also become a concern of
researchers and growers, after the realization of how limited the genetic base of
the soybean is in the country. In 1994, it was reported that for the northern and
southern North America breeding pools, there were only 19 ancestors with 17 of
them common to both regions of the US (Gizlice et al. 1994). The 19 ancestors
contributed 85% of the genes to each region. The narrow genetic base of the
crop had also been identified by earlier studies on genetic diversity in the U.S.
(Committee on Genetic Vulnerability 1972; Specht and Williams 1984). The
information reaffirmed the concept that modern soybean cultivars in the U.S.
are exceptionally uniform with the question remaining about how the impact of
selection, climate, and geography is shaping up the present-day genetic diversity
in soybean (Carter et al. 2005).
The evidence indicates that the greatest variability will be found in the center
of origin of the species, as Vavilov (1922, 1927) first established. Carter et al.
(2005) examining genetic relatedness concluded that coefficient of parentage
will increase within breeding populations derived from relatively few founding
members, thus decreasing genetic diversity. There is a definite need for con-
certed efforts to expand the genetic base of the soybean in the US. Steps in that
direction are currently taking place in several public breeding programs, parti-
cularly in the area of resistance to diseases and pests, i.e. the search for resis-
tance to brown stem rot caused by Phialophora gregata (Cianzio unpublished
information) and to soybean cyst nematode caused by Heterodera glycines (Lu
et al. 2006).
To address the possible loss of genetic diversity and the consequences due
to domestication and other factors, Hyten et al. (2006) conducted a study to
determine how human activities on the soybean species over the past 5,000 years
may have altered the DNA sequence variation in soybean. The authors
3 Soybean 65
established genetic bottlenecks in soybean as due to (1) domestication (when

humans exert artificial selection on a wild species), (2) founding (when only a
few individuals are used to introduce a crop into a new region), and (3) selection
to improve the species for cropping systems. In addition to these factors, the
reproductive physiology of the species also needs to be considered. Soybeans
are autogamous, and inbreeding in itself is predicted to decrease diversity.
The complete picture demonstrates that none of these factors favors conser-
vation of genetic diversity, unless a conscious effort is done to diminish the
effects of the mentioned factors. Hyten et al. (2006) sequenced 111 fragments
from 102 genes in four soybean populations, representing gene pools before and
after genetic bottlenecks. To represent all possible gene pools prior to and after
the genetic bottlenecks, the researchers selected for the work, elite North Amer-
ica soybean cultivars, Asian landrace founders of the elite cultivars, Asian land-
races (with no known relationship to the founding stock), and accessions of the
wild progenitor species G. soja. With these groups, they were able to show that
soybean has lost many rare sequence variants and has undergone numerous
allele frequency changes throughout its history. Despite the erosion on genetic
diversity in soybean by human intervention, the authors note that modern
cultivars have remarkably retained 72% of the sequence diversity present in
the Asian landraces, although they lost 79% of the rare alleles (frequency
0.10) found in the Asian landraces. Domestication was the human effect
that had the most important impact on genetic diversity, with less effect from
artificial selection subsequently imposed by selective breeding. These findings
suggest that the current strategy of using plant introductions in breeding with the
objective to increase genetic diversity, is justified and recommended.
Similar concerns about genetic diversity have also been shown by countries
in which soybean is a relatively new crop, such as Mexico as well as in others
where sizable collections are in place, such as India. In Mexico, in a study
conducted by Quintero et al. (2005), 24 lines developed in the country and
nine plant introductions from Brazil were characterized at the molecular level
by AFLP markers. Results indicated that: (1) lines did not group according to
geographical origin, (2) a high polymorphism level (60%) was detected, and
(3) the Mexican lines shared a high level of genetic similarity with those from
Brazil. Although the sample size used in the research did not represent all
soybeans from the humid tropics in Mexico, results indicated that the present
genetic diversity is useful to generate new cultivars. Three of the Mexican lines
were more divergent at the molecular level than foreign genotypes considered
phylogenetically different and that were used as control genotypes. In spite of
breeding and selection, and of the limited ancestors used in Mexico, there is still
genetic diversity and efforts are made to conserve it.
In India, soybean production systems in the central region of Madhya Pra-
desh, and on the Malwan plateau, require development of early maturing culti-
vars with a growing season of <90 days (Bharadwaj et al. 2007). Due to the
scarcity of early maturing cultivars, Bharadwaj et al. (2007) referred to the
National Research Centre for Soybean (NRC Soybean), in Indore to obtain
accessions and germplasm with a short growing season. Detailed data records
and subsequent analyses indicated there was considerable variation for the short-
season trait in the sub-sample of the collection studied. An interesting observa-
tion of the work was that although genotypes obtained from the same country
had a tendency to group together, the authors observed that in general, grouping
in clusters was random, indicating that geographical origin and genetic diversity
were not related. Bharadwaj et al. (2007) concluded that the germplasm identified
in the study could be used as parent material in population development to select
for early maturing lines, making use of the genetic diversity still in store.
Establishment of geographically different gene pools is an important aspect to
consider when genetic diversity of collections is assessed. Some studies have been
conducted to establish different geographical areas within countries (Dong et al.
2004; Quintero et al. 2005). Other works have been done to determine the genetic
relationship between accessions obtained from different parts of the world. This
is the case of the study by Yamanaka et al. (2007) which compared genetic
relationships between Chinese, Japanese, and Brazilian soybean gene pools as
revealed by SSR markers. The authors investigated a total of 272 cultivars from
the three countries and observed that different from previous reports (Abe et al.
2003), the gene pools from China and Japan could not totally be considered as
independent from each other. The Brazilian cultivars in turn, were distantly
related to the Chinese and Japanese gene pools, forming a separate cluster from
the two groups. On this basis it is expected that exchange of material among the
three countries would expand the genetic base and increase genetic variability.
In general, it appears that in spite of the enormous pressure exerted by both
human and physical factors on the original and natural genetic diversity in
soybean, there seems yet to be a wealth of variability that if needed could be
searched for and used. There is, however, an undeniable fact related to the
amount of effort in time and resources that may be applied to the search for the
special genetic types that the industry and consumers demand. Zohrabian et al.
(2003) have indicated the difficulties that exist in ascribing productivity gains to
specific genes or accessions because of the nature of the research process in
genetic enhancement, the relationship between genes within the genome, and
the interaction of genes with the environment. Still, the authors have deter-
mined that the lower-bound benefits from utilizing a marginal accession are
higher than the upper-bound costs of acquiring and conserving it, justifying the
expansion of the US soybean collection. And for that matter, the investments
made by any country to acquire and conserve the genetic materials of the plant
species that relate to their respective production system are worthwhile.
3.5 Major Breeding Accomplishments

The cultivated soybean has been amenable to major changes in its genetic
constitution that allowed the species to acquire wide adaptation to different
latitudes, environments, and to possess different genetic composition in the
3 Soybean 67
seed. Undoubtedly in every country where soybeans are grown, breeding is

underway to increase yield and improve other traits of economic importance.
Achievements that will be discussed here, however, pertain mainly to those
attained in the US.
When soybeans were first introduced to the US, they were mostly used as a
forage crop. Since the early 1930s major breeding efforts began to make the
soybean what it is today. Early soybean breeding in the US, was confined to
State Agricultural Experiment Stations (SAES) in Land-Grant Colleges and to
the United States Department of Agriculture’s Agricultural Research Service
(USDA-ARS) (Huffman 2004; Sleper and Shannon 2003). Legal events, i.e.,
Plant Variety Protection (PVP) Act, the subsequent PVP modification to con-
form to European cultivar protection laws and the affirmative ruling in the US
about the patenting of living material, however, stimulated the private industry
sector to invest heavily in soybean breeding. According to Frey (1996), most of
the effort in developing improved soybean cultivars or varieties presently comes
from industry, followed by soybean breeders from SAES. The USDA-ARS
spends the least amount of effort in cultivar development.
Wilcox (2001) estimated that public soybean breeders in the northern soy-
bean production region have increased seed yield about 60% over the past 60
years. Specht et al. (1999) estimated that similar progress in breeding has also
been made in the private sector, although results have been obtained during the
last two or three decades. Approximately 90% of the US soybean hectarage is
planted to cultivars developed from private programs (Sleper and Shannon
2003). Intellectual property protection, the ability to earn high returns on
research investments, and the reduction in public budgets have shifted the
majority of the soybean breeding effort from the public to the private sector.
Conventional breeding strategies have been successful in improving soybean
productivity and quality. Currently, the number of public and private cultivars
available to growers for planting is enormous, and most of them have been
developed by conventional breeding techniques. Perhaps one of the most widely
publicized achievements from the US public breeding effort has been the
commercial use of soybean lines having low linolenic acid content, developed
at Iowa State University (ISU) (Fehr and Hammond 1996, 1998). Research on
altered linolenic acid content of soybean began at ISU in 1968 after a visit by
E.G. Hammond to Unilever, a food processor industry in the Netherlands. The
researcher learned Unilever was interested in obtaining soybean oil with
reduced linolenic acid content to avoid use of hydrogenated oil in food pro-
ducts. Although linolenic acid is needed by the human body, when soybean oil is
used to manufacture margarines and other food products, it often needs to be
hydrogenated, meaning that hydrogen is added to the fatty acid chains. That
process produces trans-fatty acids, and its consumption can increase the risk of
developing cardiovascular disease when it is included in the human diet.
With these concerns in mind, two researchers from Iowa State, Hammond
and Fehr began a collaboration that spanned 30 + years and led to the devel-
opment of 1% linolenic acid soybean oil. Several patents were issued to ISU on
the account of this work, but most importantly, the new lines bred by conven-
tional breeding methods have been in commercial production since 2004–2005.
In 2004, 12,141 hectares of 1% linolenic soybean varieties were planted in Iowa
and the projection was that more than 404,686 hectares of the low-linolenic
cultivars were needed to meet the anticipated demand for oil. Asoya, an Iowa-
based corporation that manufactures oil from the low-linolenic soybeans, plans
to produce 20 million tons of oil from low-linolenic soybeans in the near future.
In 2007 three new varieties were offered to growers to enhance the production of
oil with 1% linolenic acid. Currently two manufacturers in Iowa process 1%
low linolenic soybean oil, Asoyia and Innovative Growers, the latter under the
Iowa NaturalR label.
Soybean provides about 30% of the world oil production (US Department
of Agriculture statistics 2006; www.nass.usda.gov/Publications/Ag_Statistics/
agr06), and it is mostly composed of triacylglycerols (Cardinal 2008). In the US
more than 73 million acres of soybean are grown supplying 81% of the required
edible oils and fats in the US. On average, soybean oil is composed of 110 g kg1
palmitic acid (16:0), 40 g kg1 stearic acid (18:0), 240 g kg1 oleic acid (18:1),
540 g kg1 linoleic acid (18:2), and 70 g kg1 linolenic acid (18:3). Saturated
fatty acids are not considered healthy. Unsaturated fatty acids are unstable due
to their susceptibility to auto-oxidation and production of undesirable flavors
and odors. In contrast, monounsaturated fatty acids are desirable for both
human health and oil stability (Cardinal 2008).
In 2006, the US Food and Drug Administration (FDA) began to require
food manufacturers to report on nutrition labels the amount of trans-fat in their
food products, and a healthy alternative was already in place due to the work by
the ISU team. The team identified three genes that individually reduce linolenic
acid, fan 1 in germplasm line A5 (Hammond and Fehr 1983), fan 2, and fan3
(Ross et al. 2000). Individually each of the genes would produce genotypes that
would possess an average of 2.9–4.9% of linolenic acid. The three genes com-
bined in the same genotype, however, were able to reduce the linolenic acid in
the oil to approximately 1%. The original genotypes possessing reduced lino-
lenic acid content were, however, poor looking from the agronomic point of
view. Plants were also short in height, had few pods, thick stems difficult to dry
for appropriate harvest, were sickly looking, and lodged badly (Cianzio unpub-
lished results).
Research advances to obtain the high-yielding and low-linolenic lines began
in earnest when the team at ISU developed the germplasm line A5 (Hammond
and Fehr 1983). The line was selected from the progeny of soybean mutagenized
with ethyl methanesulfonate (EMS). An intense all year round effort began
using mainly the research facilities that ISU has in Puerto Rico (Cianzio
unpublished information). In Puerto Rico it is possible to obtain two genera-
tions from October to May (Cianzio 1985), with the third generation of the year
grown in Iowa. The majority of the crossing for the release of the low linolenic
lines was conducted in Puerto Rico during the winter, along with generation
advances. Continuous cycles of crossing among mutant lines, oil and fatty acid
3 Soybean 69
analyses of each line, and subsequent selection for low linolenic content both in
Puerto Rico and Iowa were carried out in the fall and winter seasons in Puerto
Rico. Selection for agronomic traits was always conducted in the plantings at
Iowa during summer. Crossing in Puerto Rico was difficult, since A5 and lines
low in linolenic acid behaved in unpredictable ways in terms of time to flower;
pollen production was also poor (Cianzio unpublished information). To
improve high-yielding potential of the lines possessing the low linolenic acid
content, the backcross method of breeding was also utilized by conducting
cycles of backcrosses to high-yielding genotypes as recurrent parents along
with oil and fatty acid analyses of the progeny, to identify the lowest segregants
for linolenic acid in the progeny of each generation. The research effort that
lasted almost 40 years and was a real team effort, has yielded the low linolenic
acid – high yielding lines that are in commercial production today.
Another major accomplishment at the commercial level that has transcended
the physical limits of the species was achieved by the Monsanto Company with
the development of Roundup Ready1 (RR) soybean, which is the product of
genetic transformation considered a genetically modified organism (GMO).
This is one of the best examples of the use of a recently developed technology
and its applicability for product development. The technique and commercia-
lization of the RR soybean has been so successful to Monsanto, that in Feb-
ruary 2007, the Monsanto Company announced the availability of a second
transformation event, referred as RR event two or Roundup Ready2yield1.
The RR soybean from event one was among the first transgenic crops to reach
the market (Parrot and Clemente 2004). It was first commercialized in 1996, and
by 2000, RR soybean was grown on 54% of the soybean hectarage in the US.
RR soybeans are resistant to the herbicide glyphosate, the active ingredient in
the commercial herbicide Roundup, also produced by Monsanto. Presently,
RR soybeans are grown on 95% + of the hectarage in the US and in other
countries, i.e. Brazil and Argentina. RR soybeans brought more effective weed
control into the management practices of low income growers, and soybean
yields were expected to be equal or higher to conventional cultivars (Huffman
2004).
The yield level of RR soybean compared to conventional cultivars, however,
is still a debatable issue. It seems that at least in some conditions and some
environments, RR soybeans pay a yield penalty. It is argued, that the RR event
two will be free of this limitation. Growers find the technology easy to apply,
not timing critical and effective in a 2-year crop rotation. These may be some of
the reasons for the commercial success of planting RR soybeans in the US. For
a detailed description of the transgenic procedure used by Monsanto, the reader
is referred to the publication by Parrot and Clemente (2004).
The commercial use of GM soybeans has brought to the discussion table
different groups with opposed views about the use of genetically engineered or
GMO soybeans for food production. Some consider the use of GMO as one of
the greatest inventions since the beginning of farming (Huffman 2004). Others
caution that the technology has not been sufficiently evaluated as far as the
consequences for humans who consume such food products. The growing and
almost permanent controversy over GMO food products and consumers’
attempts to make better food purchasing decisions have stimulated interest in
food labeling and identity preservation. This controversy has precluded wide
use of other GMO soybean cultivars, particularly in Europe.
A second product in commercial production is a high oleic (HO) soybean
developed by DuPont, Wilmington, DE (Parrot and Clemente 2004). An extra
copy of a gene was inserted in the soybean genome resulting in gene silencing.
As a consequence, oleic acid accumulates because it is not converted to linoleic
acid, and the seed has a higher oleic acid concentration. Other transgenic
soybeans have been developed by Aventis in Strasbourg, France. In each of
the two cases, however lack of approval by the European market has prevented
wide commercial use of the transgenic soybeans. Still, it is important to recog-
nize that these are breeding accomplishments that would not have been possible
without development of the transgenic soybeans and transformation techni-
ques. Soybean breeders were also the other important component of this
collaborative work, who contributed to develop the final commercial product.
In addition to the individual case-accomplishments discussed, and consider-
ing the soybean commodity as a whole, maybe the most important breeding
achievement of all has been the development of the soybean cultivated species as
one of the most important oil crops in the world. In the US, after the initial
introduction of 19 genotypes both in the northern and southern regions (Gizlice
et al. 1994), breeding methods were used to develop numerous cultivars, first as
products of the public sector, and lately from the combined efforts of both
public and private sectors. These advancements can fairly be attributed to the
concerted effort of soybean breeders, and to the research they conducted to
develop the array of adapted cultivars available to growers.
Other breeding achievements also of similar importance to the soybean
commodity may not have had the direct commercial impact of some of the
individual cases previously discussed. However, the search for novel genes in
the National Soybean Germplasm Collection has identified new sources of
resistance to several diseases and pests, i.e. soybean cyst nematode (Lu et al.
2006), Phytophthora root rot (Burnham et al. 2003; Sandhu et al. 2005); and to
physiological traits such as drought tolerance (Carter et al. 2004) among many
others. The genes are being incorporated in new cultivars, and will produce an
impact in soybean yields through the protection and improved ability of the
cultivars to withstand biotic and abiotic stress factors. The search for yield
genes in the collection is an undergoing effort which will eventually result in new
findings. The caveat with these searches is that once the new genes are identified
and characterized, they will have to be bred into high-yielding genetic back-
grounds, before their impact in commercial production can be finally assessed.
Immediate results will not be available; however their contributions to soybean
production will be noticed and valued.
Advances in the area of molecular technology are also contributing to
breeding achievements. Presently, disease resistant germplasm lines have been
3 Soybean 71
released in which molecular markers confirm the phenotypic reactions of

resistant genotypes (Cianzio et al. 2002, 2006a, b, c, 2007a, b). Proprietary
issues and the patents of complete regions of some soybean linkage groups
(Webb 1999, 2000), have somewhat precluded a more generalized use of mar-
ker-assisted-selection as a means to increase efficiency of breeding efforts.
These patents (Webb 1999, 2000) have mainly affected the breeding for brown
stem rot and soybean cyst nematode resistance.
The future of soybean breeding will undoubtedly be greatly impacted by
advances in the area of soybean genomics. These advances will open up new avenues
for breeding, increasing efficiency and feasibility of transferring the required genes
to further improve the commercial attractiveness of the soybean. Gene discovery
stemming from structural and functional genomics research will certainly lead to
new products and to cultivars with improved nutritional and agronomic characters
(Shoemaker et al. 2003; Stacey et al. 2004). New levels of increased yield will
continue to secure the commercial success of the novel soybean types.
3.6 Current Goals of Breeding

Soybean breeding programs that include oil improvement among their objec-
tives focus on two overall goals: (i) increasing total seed oil concentration and
(ii) modifying fatty acid composition of the oil.
3.6.1 Seed Oil Concentration
Improving seed oil concentration has been an intentional or unintentional goal of

soybean breeders for centuries. The domesticated crop arose from ancestors with
lower oil content and higher protein content, as small, black, hard seeds with low
yield. Over many generations of breeding, selections for yield, agronomic char-
acteristics, and seed quality led to large yellow seeds with typical averages of 20%
oil and 40% protein making the valued global commodity of modern day.
An intrinsic positive correlation between seed oil concentration and yield is
well known (Burton 1987), and gains in oil are typically realized at the expense of
loss in total protein concentration. Modern breeders understand that the soybean
commodity is valued for its composite components of high protein soy meal and
versatile vegetable oil. Thus, a balanced approach for modest gains in oil and
yield are often targeted without substantial loss in protein concentration.
3.6.2 Fatty Acid Modification

In the US, the United Soybean Board (USB) has supported a program in recent
years known as the Better Bean Initiative (BBI), with aggressive goals to
improve soybean oil and meal quality. Oil quality improvement is targeted with
reduced saturates and elimination of trans-fatty acids produced by hydrogena-
tion. A third goal is to improve oleic acid concentration for improved oxidative
stability of the oil.
3.6.3 Reduced Saturates
Health conscious consumers currently favor a diet lower in saturated fats.

Saturated fatty acids are major dietary components responsible for elevating
cholesterol. Soybean contains two saturated fatty acids, 16:0, and 18:0. Palmitic
acid is the primary saturated fatty acid that is a health concern as it has been
correlated to cardiovascular disease. Dupont et al. (1991) suggested keeping
saturated fatty acids in the human diet below 10% on a daily basis. The US
Food and Drug Administration (FDA) advises keeping total saturated fat less
than 7% in the daily diet. Breeders working with the BBI are seeking to develop
high yielding soybeans with <7% total saturates.
3.6.4 Increased Saturates
Although reduction in saturates is a key oil quality breeding target, some

breeders pursue projects to increase saturates for the solid fat industry for tub
margarine production. Soybeans with increased 16:0 and 18:0 have favorable
processing applications to produce low trans-fat margarines (List et al. 1995;
Neff et al. 1999). An important consideration in future breeding efforts is that
unlike 16:0, 18:0 has been show to either reduce or to have no effect on serum
cholesterol levels in humans (Kris-Etherton and Yu 1997).
3.6.5 Increased Monounsaturates
Soybean oil contains the monounsaturated fatty acid 18:1. Major gains in
oxidative stability of the oil can be achieved if 18:1 concentration can be
enhanced to greater than three times that of normal soybean. The BBI target
for soybean 18:1 concentration is 65–75% of total lipid. Levels as high at 80%
18:1, further enhancing oxidative stability, have been achieved by DuPont Co.
through genetic engineering, as previously mentioned (Hitz et al. 1995).
3.6.6 Trans-fat Reduction

Global concerns with trans-fat health issues have prompted major processors to
improve engineering processes and find oilseed feedstocks capable of producing
reduced or zero trans-fat food products. In January 2006, the FDA required US
3 Soybean 73
food labels to list the levels of trans-fat, prompting intense competition and
formulation changes in the food industry. Major metropolitan areas, such as
New York City recently instituted trans-fat bans in restaurants. Soybean oil is
readily available and has been an affordable mainstay for food processors.
Reductions in soybean 18:3 concentration provide opportunities for reduced
trans-fat food products.
3.6.7 Increased Polyunsaturates
Industrial applications for inks and drying oils would benefit from increased
18:3. This would require development of a specialty soybean, as the majority of
the oil is processed for vegetable oil food applications favoring reductions in
18:3, aimed to very specific commercial markets.
3.6.8 Increasing Nutraceuticals in Seed

Soybeans have been consumed in Asian countries for centuries, and more
recently in the West, as an important source of protein. During the past few
years, a significant body of medical research has accumulated, which recognizes
soybean as having a role in the prevention and treatment of chronic diseases
(cancer, heart disease, kidney disease, osteoporosis). These effects have been
attributed to a large extent to the phytochemicals called isoflavones, which
occur naturally in soybean seeds and soy products. Recent studies have uncov-
ered substantial differences in isoflavone levels among Canadian soybean vari-
eties (Primomo et al. 2005a; Al-Tawaha and Seguin 2006).
The elucidation of the mode of inheritance of total as well as specific
isoflavone contents is necessary to design an efficient and cost-effective breed-
ing strategy for developing high isoflavone soybean varieties. The existence of
12 isomers, the complexity of the phenylpropanoid pathway by which they are
synthesized and the high, often prohibitive cost of analysis using high perfor-
mance liquid chromatography (HPLC) analysis have hampered such studies.
Recent studies conducted at the Southern Illinois University (Njiti et al. 1999;
Meksem et al. 2001) have reported the finding of several quantitative trait loci
(QTL) on three molecular linkage groups associated with different isoflavones
in the Southern soybean germplasm. In a number of studies, the Southern, later
maturing, soybean gene pool has been found to be substantially genetically
different from the Northern, short-season one, which is used in Northern US
and Canada. Recently, the first report on the mapping of QTL in Northern
soybean germplasm demonstrated that the loci and alleles in this germplasm
pool may be different from those in the Southern one (Primomo et al. 2005b)
This provides an opportunity for breeders to develop novel isoflavone profiles
by combining the Southern and Northern germplasm.
Tocopherols have been associated with several health benefits, including a

decrease in the incidence of prostate cancer among the subjects receiving a-
tocopherol (vitamin E) compared to those not receiving it; gamma-tocopherol,
found mostly in soy-based foods, appeared to promote prostate health by enhan-
cing the effects of a-tocopherol and selenium (Fischer et al. 2001). In fact, it was
suggested that gamma-tocopherol, which is the most prominent in soybean seed
may have greater antioxidant benefits than its cousin, a-tocopherol (Rajcan et al.
2005).
Unlike maize tocopherols, which have recently been studied genetically (Wong
et al. 2003), there is virtually no information on the genetic control of tocopherols
in soybean at all. We have recently completed a study using a RIL population
from a cross between parents with different tocopherol levels and profile to study
the inheritance and map QTL for the trait in soybean seed (Wohleser 2006;
Rajcan unpublished information). Considering the high market value of toco-
pherols, it is conceivable that breeding of lines with elevated levels of tocopherols
may open new market opportunities for the soybean industry. Soybean breeding
programs addressing this objectives will need to be developed.
3.7 Breeding Methods and Techniques
3.7.1 Gain from Selection
Soybean seed yield continues to increase in many of the world’s soybean produc-
tion areas. Soybean breeders have periodically evaluated genetic gains by grow-
ing soybean cultivars released over a period of time in common trials to quantify
annual genetic improvements. Generally linear regression is used to estimate
annual rates of gain. From a number of studies of northern US and Canadian
cultivars, annual gains were estimated to be from about 10 to 30 kg ha1 (Boerma
1979; Luedders 1977; Specht and Williams 1984; Voldeng et al. 1997; Wilcox et al.
1979). In examining sixty years of public cultivar development using cooperative
test results (Uniform Soybean Tests, Northern Region), annual improvement
rates were found to range from about 22 to 30 kg ha1 (Wilcox 2001). A study of
genetic progress using southern US soybean cultivars reported an annual
improvement rate of 14 kg ha1 (Ustun et al. 2001). Similar studies in India
(Karmakar and Bhatnagar 1996) estimated an annual improvement rate of 22 kg
ha1. Specht et al. (1999) also collected data from multinational private soybean
breeding companies for maturity group II and III privately developed cultivars
and reported an annual improvement rate of about 30 kg ha1 which was triple
the rate from their previous report using publicly developed cultivars (Specht and
Williams 1984). Long term yield trends provide another avenue to explore
soybean improvement. Specht et al. (1999) reported that US soybean yields
increased at approximately 23 kg ha1 annually over the period from 1928 to
1998. In the Midwest USA, during the period 1972 to 2003, county yield increases
ranged from about 15 to 38 kg ha1 annually (Egli 2008). Significant yield
3 Soybean 75
increases were not seen in a number of countries growing non-irrigated soybeans

nor in counties where high levels of double crop soybean were grown (Egli 2008).
When long term yield trends are examined, the yield improvements seen are a
result of improved agronomy and production practices, environmental changes
and improved genetics. Similarly to maize (Kucharik 2008), earlier planting of
soybean is also likely responsible for some of the increased yield seen over time.
The rising concentration of atmospheric CO2 also plays a role in annual
increasing yield. During the period from 1972 to 1997, it has been estimated
that about 15% of on-farm soybean yield increase is due to increased atmo-
spheric CO2 (Specht et al. 1999). To determine the role that genetics plays in
improved yield, examination of old and new cultivars in the same trials may be
the most useful methodology.
3.7.2 Sources of Gains from Selection
From studies of short-season soybean in Canada, newer cultivars had smaller leaf
area indices accompanied by increased leaf photosynthetic and stomatal con-
ductance rates compared to older cultivars (Morrison et al. 1999). Yield improve-
ments in newer cultivars have been the result of higher harvest index manifested
in a greater number of seeds per plant (Morrison et al. 2000). Yield improvements
in China were also found to be primarily due to increases in harvest index (Cui
and Yu 2005). In a comparison of two older and two newer cultivars both an
increase in harvest index and an increase in dry matter accumulation explained
the yield improvement; however in this comparison, harvest index was less
important than dry matter accumulation (Kumudini et al. 2001). Decreased
lodging in newer cultivars has been reported in a number of studies (Specht
et al. 1999; Ustun et al. 2001; Voldeng et al. 1997; Wilcox 2001), which may
play a role in improved yield through maintenance of an upright, photosynthe-
tically optimally-oriented canopy, ease of harvesting and reducing harvest losses.
Stress tolerance in maize has increased in more modern cultivars (Tollenaar and
Wu 1999). In soybean, as plant populations increased from 33 to 100 plants m2,
differences between older and new cultivars became more apparent (Specht et al.
1999). Similarly, genetic improvement rates were low at low populations and were
maximized at plant stands of three to four times current commercial plant stands
(Cober et al. 2005). Selection for higher yield may have indirectly contributed to
selection for tolerance to stress resulting from higher plant populations.
3.7.3 Parent and Population Structure
Soybean breeders need to choose the type of cross, or the parental and popula-
tion structure to use in hybridization for population development. Choices
range from the simple biparental cross to complex crosses or even the case
where strict pedigree information is lost to favor recombination as in recurrent
selection programs. To provide a snapshot of current practices of public soy-

bean breeders, an analysis of cultivar and germplasm registrations in Crop
Science, and cultivar descriptions in the Canadian Journal of Plant Science
was undertaken with parent and population structure being summarized
(Table 3.1). Publicly developed cultivars were overwhelmingly derived from
biparental crosses. A very limited number of cultivars were developed from
backcross-derived populations. In this case, the two introgressed traits were
modified oil composition, and disease resistance. In the development of germ-
plasm lines, more complex parental structures were used, although biparental
crosses were still used about 80% of the time. Three way crosses were used to
develop germplasm with modified oil composition, pest resistance and dense
pubescence. Backcrosses were used to introgress pest resistance, and growth
habit traits into established cultivars.
Table 3.1 Summary of soybean cultivar and germplasm descriptions published from 2004 to
2006 in Crop Science and Canadian Journal of Plant Science
Cross type Breeding method1
Bi-parental Three way Back-cross SSD2 Pedigree Bulk/Mass EGT3
Cultivars
Crop Sci. 30 0 2 24 2 0 5
CJPS 10 0 0 8 1 1 0
Total 40 0 2 32 3 1 5
Germplasm
Crop Sci. 33 3 6 8 6 0 14
1
It was not possible to determine the breeding method from all descriptions.
2
Single seed descent or a modification of single seed descent.
3
Early generation testing.
Parents may be selected from publicly or privately developed cultivars,

released germplasm lines, plant introductions or unreleased breeding lines. In
the US, it is increasingly common to find soybean cultivars with patent protec-
tion (Table 3.2). In the period from 1991 to 1995 only four cultivar patents were
Table 3.2 Number of patents for different crops issued by the United States Patent and
Trademark Office resulting from a search for cultivar, variety, or hybrid, or inbred in patent
titles
Crop 1986–1990 1991–1995 1996–2000 2001–2005
Wheat 0 0 0 7
Canola/rapeseed 0 0 0 10
Canola/rapeseed inbred 0 0 0 0
Corn/maize 0 5 103 93
Corn/maize inbred 6 63 308 263
Soybean 0 4 287 388
3 Soybean 77
issued for soybean by the US Patent and Trademark Office, while ten years later
388 patents were issued during the period 20012005. The number of patents
issued in the later period approximately equals the number issued for maize,
and far exceeds the number issued for rapeseed/canola or wheat. A concern
regarding variety patenting is the limit imposed on use of patented cultivars as
parents. This limitation in use is commonly included in the patents issued.
Soybean breeders in public and small or medium private institutions may
have fewer unencumbered choices for parents in the future.
3.7.4 Advancing Toward Homozygosity

Following a successful cross and development of heterozygous plants, the
soybean breeder needs to return the breeding material to the homozygous, or
more accurately the near homozygous state. Breeders have two basic choices,
rapid fixing of genes in soybean lines usually without any selection as it is done
in the single-seed descent method, or practicing selection during the selfing
generations. From an analysis of recent cultivar and germplasm registrations
in Crop Science, and of cultivar descriptions in the Canadian Journal of Plant
Science, single seed descent was the preferred breeding methods of public
soybean breeders (Table 3.1). Pedigree breeding methods, with or without
early generation testing, was a distant second choice of these breeders. The
bulk method, with or without mass selection, was rarely used. When examining
germplasm releases, a greater range of breeding methods were reported.
Single seed descent can be rigorously applied, where each pure line is developed
from a different F2 plant, since only a single seed is selected and advanced to the
next generation. To save labour, the single seed descent method can be modified;
single pods may be harvested and all of the seeds in the pod are advanced to the
next generation. This modification is identified as single pod descent or modified
single seed descent. To determine the effect of multiple seed descent resulting from
generation advance of a pod rather than a single seed in grain legumes, a simula-
tion study predicted that the practice should still result in every third F6 plant being
derived from a different F2 plant (Macchiavelli and Beaver 2001). A further
modification introduces selection against inferior genotypes during single pod
descent. Single pod descent with selection, in this case for maturity, was found
desirable since it reduced the size of the population advanced and produced
superior lines (Destro et al. 2003). In a similar study pedigree selection, single
seed descent, and single seed descent with selection for early maturity were com-
pared, and single seed descent with selection was found to be the most cost-
effective (Byron and Orf 1991). This modification of single seed descent would
only be useful when generation advances are grown in environments where selec-
tion can be practiced and where the trait is highly heritable on a single plant basis.
Early generation testing is a procedure which tests heterogeneous families
and then selects homozygous lines from superior families. The procedure gains
efficiency since it results in yield tests of fewer homozygous lines. In an evalua-

tion of possible variants of early generation testing, F2 derived families were
suggested as the most efficient testing generation compared to F1 or F3 derived
families (St. Martin and Geraldi 2002). Single rows can be used to yield test in
early generations since they were good predictors of performance in multiple
environment advanced yield trials (Hegstad et al. 1999). In an evaluation of
optimum selection pressure for early generation families versus preliminary
pure line testing, historical data from the Ohio State soybean breeding program
was examined. An equal selection intensity was recommended across these
stages of a breeding program (St. Martin and Futi 2000).
3.7.5 Participatory Plant Breeding

Participatory plant breeding is a new model for plant breeding which, while
primarily focused on increasing the productivity of resource-poor farmers in the
developing world, could be used throughout the agricultural sector. Most of
this research is conducted in farmers’ fields with farmers and researchers work-
ing side by side. Much information on participatory plant breeding has been
reviewed and synthesized in a monograph by Weltzien et al. (2003). Beside the
social benefits of participatory plant breeding, such as empowering citizens in
rural communities, benefits in breeding may include increased productivity for
niche producers in niche environments. Farmer participation is included to
better understand farmer preferences and to meet end-user needs. Participatory
plant breeding could be most beneficial where major crop commodity research
is unavailable (orphan crops), where marginal production areas, or high geno-
type by environment interactions preclude development of a few widely adapted
cultivars, or where crops have diverse end uses.
Most participatory plant breeding programs involve farmers testing geneti-
cally fixed, i.e. pure line materials. Many programs also involve farmers in
setting plant breeding objectives while fewer programs involve farmers in
selection within segregating generations. With the exception of on-farm county
strip trials, which may include public or private experimental lines, to our
knowledge there are no reports of participatory soybean breeding programs.
On the basis of results from other crops (Kornegay et al. 1996), soybean
breeders may find occasions where it would be advantageous to adopt partici-
patory plant breeding strategies.
3.7.6 Selection Among Pure Lines

Selection among breeding lines approaching ‘pure line status’ or homozygosity
begins with discarding inferior lines and ends with identification of a few
superior lines that are moved to commercial production. Breeders need to
3 Soybean 79
allocate resources among and within populations as well as within years of

testing from preliminary to advanced trials.
From an evaluation of 30 lines in each of ten populations, it was recom-
mended that a maximum of 15 lines per population should be tested to allow
sampling of as many populations as possible (Helms et al. 2002). To evaluate
resource allocation for preliminary trials, five test locations were used to verify
selection based on either one or two locations in preliminary tests, and the use of
a single replicated test at a single location (Helms et al. 2002) was recommended.
To deal with large preliminary trials and the spatial variation that can be
present within these trials, a number of statistical approaches are available.
Nearest neighbor analysis or lattice designs were found more efficient, com-
pared to randomized complete block designs, in reducing error variance in
soybean experiments (Vollmann et al. 1996). Helms et al. (1995), however, did
not find nearest neighbour adjustments useful compared to unadjusted means
for yield selection in two populations.
3.7.7 Intra-cultivar Variation
While cultivars of self-pollinating crops are regarded as pure lines or highly

homogeneous, there is some suggestion that variation within cultivars could be
exploited as suggested by Rasmusson and Philips (1997) in barley and by
Tokatlidis et al. (2004) in wheat. In soybean, three cultivars were studied for
intra cultivar variation for seed composition, seed oil and protein levels, and
significant differences were found among lines within each cultivar for seed
composition (Fasoula and Boerma 2005). A further examination of the three
cultivars found intra cultivar variation for seed weight and time to maturity
(Fasoula and Boerma 2007). The authors suggest that intra cultivar selection
could be carried out to retain uniformity within cultivars or that the newly
uncovered variation could be used for further improvements.
3.7.8 New Technology in Plant Breeding Operations

Technological advances continue to be made in areas that affect plant breeding
operations. Gains in efficiency allow more plots to be grown with the same
amount of personnel or diversion of personnel to new areas of research.
Software programs, either commercially developed or written by the indivi-
dual breeder, are used throughout breeding programs. Tasks managed by soft-
ware include seed inventory, experimental design, field planning, generation of
field books, data logging, experimental analysis, and multiple location and year
analysis. A major improvement involves the use of relational databases com-
pared to flat file systems. Flat file systems involve separate files for each
experiment and a simple task such as a name change is required to be made in
each separate file; while in a relational database, a single name change results in
the name being changed in all locations throughout the system. Laboratory
information management systems (LIMS) are software programs which man-
age samples, instruments and dataflow. Near-infrared reflectance spectroscopy
instruments, balances, and other data collection devices can be interconnected
which reduces chances for errors in data transcription and aids in data collec-
tion efficiency.
Data management has been improved through less direct entry of identifiers
or data observations. Postal equipment can be used for direct printing on seed
envelopes or bags. Bar code readers can be used to directly enter plot numbers
from bar codes on bags or tags. As well bar code readers can be used to enter
data when data options are preprinted on summary sheets, for example, hilum
colours are entered through an operator ‘shooting’ the appropriate hilum
colour bar code. Radio frequency identification (RFID) tags can replace bar-
codes and barcode readers. RFID pot labels are available and used in the
horticultural industry. RFID tags are being tested on individual seed packets,
as a replacement for barcodes, where all seed envelopes in a shipping box could
be read by placing the box near a RFID reader.
Improvements in harvesting technology allow faster harvesting and reduced
post-harvest processing. Combine harvesters are available which can harvest
and yield test individual progeny rows and make a selection or rejection
decision based on predetermined parameters. Small plot combines can be
equipped with on board weighing systems, as well as on board near-infrared
equipment to determine moisture, protein and oil content. Plot combines can
also be equipped with samplers which will sub-sample the plot harvest, then
package and label the sample. Developments in in-crop canopy scanning tech-
nology may allow prediction of yield before harvest because of the significant
relationship between normalized difference vegetation index (NDVI) at pod
development and early seed filling and seed yield (Ma et al. 2001). This technol-
ogy could allow rejection of lines before harvest and reduce use of resources for
harvesting.
3.8 Integration of New Biotechnologies into Breeding Programs
The accelerated production of biodiesel in North America has prompted

greater attention by breeders to target increases in total crude oil per hectare
of soybean production. Pantalone et al. (2004) outlined a series of quantitative
trait loci (QTL) that govern seed oil content. Some of those QTL are envir-
onmentally stable, while others are sensitive to particular types of environmen-
tal growing conditions. However, few of the oil QTLs have been confirmed.
Opportunities exist to confirm oil QTL to facilitate use of this biotechnology for
soybean breeders for increasing total oil. Moreover, examining the impact that
accumulation of environmentally stable oil QTLs have on seed oil
3 Soybean 81
concentration would make further contributions to existing knowledge. This

information, together with measurements of soy meal characteristics will lead
to opportunities to develop new varieties more suitable to biodiesel production
in major soybean producing regions, while maintaining the quality of the soy
meal for livestock and human nutrition industries.
Recent discoveries in molecular markers will help direct strategies for
improving the quality of soy oil with respect to altered fatty acid profiles.
Those discoveries are inherently based upon germplasm developed for modified
fatty acid composition.
3.8.1 Reduced Saturates – Germplasm and Biotechnologies

The germplasm line N79-2077-12, developed by USDA-ARS, Raleigh, NC was
the first low 16:0 soybean (Wilson 2004; Burton et al. 1994). It has the recessive
genotype fapncfapnc which reduces the C16:0 content to about half that of
normal soybean. The same level of 16:0 reduction was found in the germplasm
ELLP2 which has the fap*fap* genotype. A different allele is found in germ-
plasm C1726 which has the genotype fap1fap1 which does not reduce the 16:0 as
effectively as the other two alleles. However if fap1fap1 is coupled with fapncfapnc
as in the case of both N94-2575 and C1943 germplasm lines, then the collective
reduction of 16:0 is about one-third that of normal soybean. This low level of
palmitate can alternatively be realized by a different single allele fap3fap3 as
occurs in the germplasm A22 (Wilson 2004). Breeders have assembled various
combinations of these alleles to more modern adapted germplasm and elite
lines.
Cardinal et al. (2007) demonstrated that the low 16:0 phenotype fapnc was
due to a deletion in the GmFATB-1A gene. The authors developed allele-
specific PCR primers for this gene, and they also showed that on average,
there is a yield reduction when a line is homozygous for the fapnc allele.
Allele-specific primers are perfect matches between the DNA marker and the
existence of the trait. Such markers are highly valuable to breeders who can
develop larger populations with the intent to find high yielding types among the
group of individuals that carry the specific DNA marker. Moreover, breeders
who are successful in combining high seed yield with low 16:0 have unique
genetic resources that can be further utilized for oil improvement (see major
breeding achievements section, above).
3.8.2 Increased Saturates – Germplasm and Biotechnologies

Wilson (2004) reported that germplasm homozygous recessive for fap2 (C1727),
fap2b (A21), fap4 (A24) or fap5 (A27) produce oil with 150% or greater 16:0 than
normal soybean. Stearic acid concentration in soybean may be genetically
increased by mutations at the Fas gene locus (Spencer et al. 2003). Most of
these variants have been induced by chemical or X-ray mutagenesis. An
exception is the USDA germplasm line, FAM94-41 (9% C18:0) which
carries a natural mutation, temporarily designated as the recessive fasnc
allele (Pantalone et al. 2002). Spencer et al. (2003) detected a major QTL
on linkage group B2, near the Fas locus governing increased 18:0 concen-
tration from FAM94-41. Five additional germplasm lines were reported to
carry homozygous recessive alleles: fasa [A6, (Hammond and Fehr 1983)],
fasb [FA41545, (Graef et al. 1985)], fas [A81-606085, (Graef et al. 1985)], st1
[KK-2 (Rahman et al. 1997)] or st2 [M25, (Rahman et al. 1997)] that
influence 18:0 concentration in soybean oil. Increases in 16:0 and 18:0
would be of benefit for the margarine industry in order to produce low
trans-fat tub margarine.
3.8.3 Increased Monounsaturates – Germplasm and

Biotechnologies
The germplasm line N78-2245 was the first soybean developed with elevated
C18:1 (approximately 42%), nearly twice that of normal soybean (Wilson
2004). This line contains a natural mutation in the FAD2-1 gene encoding
the predominant o-6 desaturase. The FAD2 gene was clearly shown to
govern soybean 18:1 concentration. Hitz et al. (1995) expressed it in anti-
sense orientation, producing up to 80% 18:1. Also, a soybean line with
natural gene combinations was achieved with >70% 18:1 (Burton pers.
comm.). Recently Burton et al. (2006) registered the germplasm line N98-
4445A (60% 18:1) in the USDA active germplasm collection, making it
freely available to breeders targeting improvement in 18:1 concentration.
Breeders working with the BBI embarked on a challenging mission over the
last few years to rapidly develop new cultivars with elevated 18:1 content.
The goal is for breeders to utilize the new USDA germplasm line, N98-
4445A (Burton et al. 2006) and introgress six QTL loci that collectively
govern mid-18:1 (50–65%). QTL are genomic regions genetically linked to a
trait, and typically several QTL (several genes) need to be accumulated
together in a single individual for full trait expression. In the mid-oleic
soybean project, the goal is to assemble three QTL in a backcross line
and three complementary QTL in the same backcross line and make a
convergent cross after the BC5 stage. These six QTL are represented by
simple sequence repeat (SSR) markers in the plant DNA. Additional work
led to the discovery of single nucleotide polymorphisms (SNP) associated
with the mid-18:1 trait. These SNPs are single base-pair changes in the
DNA, and as SNP maps are developed and refined (Choi et al. 2007), this
new technology will serve as an excellent selection mechanism to improve oil
quality.
3 Soybean 83
3.8.4 Reduced 18:3 – Germplasm and Biotechnologies
A major commercial thrust targets low C18:3 soybean development to meet

the demands of reduced trans-fat vegetable oils and food products. The first
reduced 18:3 germplasm line was N78-2245 with 6% 18:3 (Burton et al. 1989)
which was produced through recurrent selection for increased 18:1 concentra-
tion. Wilcox et al. (1984) used EMS to create a recessive fanfan C1640
germplasm line with approx 3.5% 18:3. Germplasm line A5 was also created
by chemical mutagenesis producing the fan1fan1 genotype, conferring
approximately 4% 18:3 (Hawkins et al. 1983). Fehr et al. (1992) were able to
combine two additional mutations forming germplasm line A29 with three
recessive alleles fan1fan1fan2fan2fan3fan3. This genetic material fostered a
recent discovery (Bilyeu et al. 2003) of three different isoforms of omega-3
desaturase genes. This lead the authors to develop ‘perfect’ molecular markers
designed from the actual DNA sequence, which allows molecular breeders to
use DNA to select individuals carrying one or more of the low 18:3 genes with
complete accuracy. Accumulation of the three recessive genes collectively will
reduce 18:3 concentration in soybean oil to 1%. Plans are currently in pro-
gress for a USDA release of a germplasm line containing two publicly avail-
able recessive alleles (Bilyeu pers. comm.). New commercial soybean cultivars
containing all three recessive alleles are currently in production (Fehr pers.
comm.).
3.8.5 Increased Polyunsaturated Fatty Acids – Germplasm and

Biotechnologies
Soy oil components are routinely used in inks, coatings, and resins. Increasing
the C18:3 concentration would enhance these industrial products. Currently no
improved germplasm have been reported with elevated 18:3. However the
USDA germplasm collection contains more than one hundred accessions of
Glycine soja, the wild relative of soybean, that exhibit 18:3 concentrations more
than twice that of cultivated soybean (Glycine max). The wild species is fully
sexually compatible with soybean, allowing gene transfer for enhanced poly-
unsaturated fatty acids to be realized. Pantalone et al. (1997a) made interspe-
cific hybridizations between N87-2120-3 (G. max) x PI342434 (G. soja) and
PI424031 (G. soja), identifying a wide range in o-6 desaturation and o-3
desaturation among progeny, suggestive that the wild species carries alternative
forms of Fad and Fan alleles. The recombination of alternative desaturases with
G. max alleles acted in an additive genetic manner, producing higher 18:3
concentration in the oil (Pantalone et al. 1997b; Rebetzke et al. 1997). Oppor-
tunities exist to create mapping populations targeting the discovery of QTL for
unique G. soja alleles, and use marker-assisted selection specifically to target
increased 18:3 for industrial uses.
3.8.6 Oil Constituents with High Value
3.8.6.1 Sterols
The modern soybean oil refining process allows capture of stigmasterol, a
compound that is well valued for its commercial synthesis of steroidal hor-
mones and pharmaceutical products (Wilson 2004). Stigmasterol, along with
campesterol and b-sitosterol are three secondary alcohol phytosterols that are
present in soybean oil. Stigmasterol occurs in highest concentration. Although
sterol concentration in the oil of soybean seeds is positively correlated with
growth temperature, there is little variation for breeders to take advantage of.
Little is known of alleles that influence the genetic regulation of soybean sterols
and these compounds typically are not traits targeted for improvement by
breeders. Nevertheless, Wilson (2004) showed a significant negative correlation
between stigmasterol concentration and 18:3. Indeed when 18:3 dropped from
10 to 4% of lipid concentration, stigmasterol more than doubled. Thus breeders
working on developing low 18:3 lines to cultivar status may have opportunities
to verify stigmasterol concentration to document enhancement of this valuable
oil constituent for the commercial oleochemical industry.
3.8.6.2 Tocopherols
Soybean oil contains delta, gamma, and alpha forms of tocopherol. The a-
tocopherol form is natural vitamin E, and soybean is the leading commercial
source of this vitamin. Tocopherol compounds protect polyunsaturated fatty
acids from oxidation. As antioxidants, the D form is more effective than the
g form, followed by the a form of tocopherol (Wilson 2004). Despite the
presence of 1,000–2,000 mg kg1 of tocopherols in soybean oil following
the refining process, the oil still requires hydrogenation to maintain adequate
oxidative stability (Wilson 2004). This suggests that genetic improvement of
this oil constituent would augment breeders targeted oil quality goals. For
example, Wilson (2004) indicated that soybeans with genetically reduced 18:3
showed greater a-tocopherol, yet lower g-tocopherol. The decline in oxidative
stability from the lowered g-tocopherol could be offset if the increased 18:1
trait was coupled with reduced 18:3, and improved processing efficiency of
vitamin E would be a favorable constituent product.
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Chapter 4
Oilseed Rape
Wolfgang Friedt and Rod Snowdon
4.1 Introduction
Oilseed rape, or canola (Brassica napus ssp. napus; genome AACC, 2n ¼ 38) is
today the world’s third-leading source of both vegetable oil and oil extraction
meal. Total world consumption of vegetable oil amounts to approx. 97 million
metric tons (2003), of which 27.9 Mt is soybean oil, 27.8 Mt palm oil, 12.1 Mt
rapeseed oil, 8.0 Mt sunflower oil, 5.8 Mt peanut oil, and 4.9 Mt cottonseed oil.
Due to its favourable seed oil composition, low-erucic acid rapeseed or canola
oil is a valuable source of nutritional oils and fats (salad oil, margarine). For
example, from the total rapeseed oil produced and processed in Germany
(approx. 2.5 Mio. t) about 0.5 Mio. t are used for nutritional purposes. The
majority, however, is used for producing transportation fuel; around 1.5 Mio. t
are processed into biodiesel (rapeseed oil methyl ester, RME) while some
0.5 Mio. t of processed oil are directly used in diesel engines of tractors or
lorries (Thywissen, personal communication). The demand and use of vegetable
oil for non-food purposes is currently rising world-wide. A rough overview on
the major procedures for the production of biofuels from vegetable oils is
presented in Fig. 4.1 (source: http://www.biofuelstp.eu/fuelproduction.html;
see also Demirbas 2007).
The species Brassica napus L. originated through spontaneous interspecific

hybridisation between turnip rape (Brassica rapa L., syn. campestris; genome
AA, 2n ¼ 20) and cabbage (Brassica oleracea L.; genome CC, 2n ¼ 18), resulting
in an amphidiploid genome comprising the full chromosome complements of its
two progenitors. Because no wild B. napus forms are known, it is assumed that
W. Friedt (*)
Department of Plant Breeding, Justus-Liebig-University of Giessen, Giessen,
Germany
e-mail: wolfgang.friedt@agrar.uni-giessen.de

92 W. Friedt and R. Snowdon
Fig. 4.1 Schematic overview on the production of biofuels from vegetable oils (image
re-drawn from source: http://www.biofuelstp.eu/fuelproduction.html)
the species appeared relatively recently when the parental species began being
cultivated in geographical proximity. The occurrence of spontaneous chromo-
some doubling in crosses among closely-related Brassica diploid species is well
documented; the related amphidiploids Indian or brown mustard (Brassica
juncea; genome AABB, 2n ¼ 36) and Abyssinian or Ethiopian mustard (Bras-
sica carinata; genome BBCC, 2n ¼ 34) arose in the same manner after crosses of
black mustard (Brassica nigra; genome BB, 2n ¼ 16) with B. rapa and B.
oleracea, respectively.
Brassica vegetables and oilseeds were among the earliest plants to be
systematically cropped by mankind. There are indications that a vegetable
crucifer was widely cultivated as early as 10,000 years ago. In India records
have been identified which suggest that oilseed brassicas (probably B. rapa)
were being used as early as 4000 BC, and 2000 years ago their use had spread
into China and Japan. Swedes (B. napus ssp. napobrassica) were known in
Europe at the time of the Romans, and utilization (probably of B. rapa) for
oil purposes in northern Europe is thought to have begun around the 13th
century. By the 16th century, rapeseed was the major source of lamp oil in
Europe, although it was not until the 18th century that significant cultivation
areas of the crop were recorded (Kroll 1994; Kimber and McGregor 1995).
For winter oilseed rape only three distinct local landraces are known. These
evolved in different European climate zones and hence display variation in
vegetative growth and winter hardiness. The first released cultivar ‘Lembkes’,
selected in Germany from a Mecklenburg landrace in the early 20th century,
was extensively exploited in French, Swedish, German and Polish breeding
4 Oilseed Rape 93
programs. Spring-sown oilseed rape was first grown in Canada in the mid
20th century. Large-scale worldwide production of oilseed rape did not begin
until the mid 1970s, however, when the value of rapeseed oil and seed meal
was significantly improved as a result of intensive breeding efforts (see
below).
4.3 Varietal Groups
The primary gene pool of oilseed rape is separated into two distinct B. napus
subspecies, comprising on the one hand the swedes (B. napus ssp. napobras-
sica) and on the other hand B. napus ssp. napus. The latter includes winter and
spring oilseed and fodder rape forms, along with distinct leaf rape forms
(B. napus ssp. napus var. pabularia) that used to be common as a winter-annual
vegetable in many parts of the world (Siberian kale, Hanover salad; German:
Schnittkohl; French: chou à faucher; Chinese: xi yang you cai). Oilseed forms
of B. napus are cultivated in Europe and Asia predominantly as winter rape-
seed, whereby in Australia, Canada, and northern Europe only spring forms
are suitable. The differentiation into winter and spring forms is governed by a
genetic mechanism controlling the requirement for vernalisation to promote
the onset of flowering. Spring oilseed rape does not require vernalisation and is
not winter-hardy, hence the crop is sown in spring and stem development begins
immediately after germination. Winter oilseed rape on the other hand is sown in
autumn and survives the winter in a leaf rosette form on the soil surface. In the
following spring a long vertical stem develops, and shortly before the floral
development lateral branches are formed. Flowering generally occurs in late
spring, with pod development and ripening taking place over a period of around
6–8 weeks until mid-summer. In contrast to B. rapa and B. oleracea, its diploid
progenitors which often display self-incompatibility, B. napus is a facultative
outcrossing species with a high degree of self-pollination. When insect pollinators
are abundant a greater proportion of cross-pollination can occur.
4.4.1 Genetic Diversity in the Primary Gene Pool
Modern oilseed rape breeding material has a relatively narrow genetic diversity
(Hasan et al. 2006). This is attributable to a combination of geographical
constraints and selection bottlenecks during the origin of the species and its
subsequent domestication. In particular, the zero erucic acid and low glucosi-
nolate seed quality traits (00-quality) carried by the vast majority of modern
varieties (canola varieties) originate from single sources, namely the spring
cultivars ‘Liho’ and ‘Bronowski’, respectively. Consequently, there is a need
to introduce new genetic variation to breeding material, since most early

00-quality cultivars shared a more or less common parentage (Thompson
1983; Downey and Rakow 1987).
Owing to their generally unsuitable seed characters, however, in particular
high contents of seed erucic acid (C22:1), glucosinolates, and other anti-nutritive
substances, fodder and vegetable rape forms have been generally overlooked for
breeding of oilseed cultivars in recent decades. On the other hand, such material
represents a potentially valuable source for improved pathogen and pest resis-
tance, and introduction of untapped germplasm into breeding lines also has the
potential to improve heterosis in hybrid breeding. However, the construction of
distinct genetic pools, as used for example in maize hybrid breeding, is still in
progress in commercial breeding efforts.
Due to the large number of closely related cruciferous crop species an enor-
mous variety of germplasm resources are available in international gene bank
collections for evaluation and introgression of traits of agronomical interest into
oilseed Brassica breeding material. Such resources have often been successfully
used in particular for the identification and interspecific transfer of novel disease
resistances to oilseed rape breeding lines. The number and diversity of available
Brassica gene bank accessions can make it difficult to identify relevant germplasm,
however, even within the primary and secondary gene pools. Well-characterised
core collections, that represent as much as possible of the available diversity within
a manageable number of genotypes, are therefore a valuable resource for utiliza-
tion of novel germplasm in breeding efforts.
Compared to the narrow gene pool of present 00-quality oilseed rape breed-
ing material, erucic acid- and glucosinolate-containing plant material repre-
sents a comparatively genetically divergent source for the development of
heterotic rapeseed forms (see Röbbelen 1975; Thompson 1983; Schuster
1987). Because of the emphasis on oil quality, such material has found only
limited use in practical rapeseed breeding in the past few decades. However,
strong heterotic effects are observed in experimental crosses between material
of distant geographical and genetic origin (Lefort-Buson et al. 1987; Brandle
and McVetty 1990), and efforts are increasing to develop new cytoplasmic-
genetic male-sterile and restorer lines as the most promising system for the
production of new hybrid cultivars. Following appropriate quality conversion,
inbred lines and DH lines with a high genetic distance to existing 00-quality
varieties have the potential to become an important resource for the develop-
ment of high-performance pools with improved combining ability compared to
existing 00-rapeseed material.
Within the framework of a recently completed European project ‘Brassica
collections for broadening agricultural use’, extensive collections of Brassica
accessions from European gene banks were evaluated for morphological diver-
sity and selected agronomic traits (van Soest et al. 2004). Within four interna-
tional sub-groups, the available gene bank material for B. oleracea, B. rapa,
B. napus and B. carinata was documented, characterised, evaluated and ratio-
nalised to generate core collections representing as much as possible the range
4 Oilseed Rape 95
of available diversity useful for Brassica crop breeding. Passport data for over
3,500 B. napus accessions are available in the ECP/GR Central Crop Database
(BrasEDB: http://www.cgn.wur.nl/pgr/collections/brasedb/). A preliminary
core collection of 200 accessions was selected, covering the different systematic
groups, using various evaluation criteria (Poulsen et al. 2004). Particular
emphasis was placed on the geographic origin of the material, with the collec-
tion being selected to represent as much as possible the diversity present in all of
the countries for which accessions were available. Minimum descriptors for
numerous morphological characters were characterised, and together with data
from agronomical evaluations this information has been made available on the
BrasEDB website. In order to facilitate the use of the core collection for oilseed
rape breeding purposes, a number of relevant resistance and seed quality traits
were evaluated in the preliminary core collection in field and greenhouse trials.
Around 1,100 accessions were analysed for various seed quality characters, and
together the morphological, resistance and quality data were used to reduce the
core collection to around 150 accessions that cover the broad variation in
agriculturally important traits (Lühs et al. 2003; Poulsen et al. 2004). A subset
of the preliminary core collection was also genotyped using SSR markers to
evaluate the extent of genetic diversity in the oilseed-type accessions (Hasan
et al. 2006). Collectively the final core collection comprises a genetically diverse
set of genotypes with wide variation for numerous traits of agronomic interest.
Because commercial oilseed rape breeders were directly involved in the evaluation
and selection of the material, these accessions represent a valuable resource
for more detailed screening of further traits of interest with regard to introgres-
sion of novel germplasm into canola and oilseed rape breeding material (Poulsen
et al. 2004).
Whilst such collections are valuable in identifying ‘hotspots’ of variation
within the relevant gene-pools, they usually consist of heterogeneous and
heterozygous breeding material. This limits their long-term use for correlating
detailed genetic studies. To overcome these problems British researchers and
breeders are currently developing a homozygous B. napus ‘Diversity Fixed
Foundation Set’ (DFFS) for long-term molecular and trait exploration in the
oilseed rape gene-pool. The DFFS lines, fixed from founder lines by haploid
techniques or inbreeding by single-seed descent, are defined as ‘an informative
set of genetically fixed lines representing a structured sampling of diversity
across a genepool’. The fixed lines will be multiplied and archived and will be
available for distribution (for more information see http://www.brassica.info/
resource/plants/diversity_sets.php/).
4.4.2 Expanding Genetic Variability by Interspecific Hybridisation
One strategy to broaden the genetic basis of oilseed rape breeding material is the
production of resynthesised (RS) rapeseed by crossing the original ancestors,
B. oleracea and B. rapa. This has the potential not only to increase genetic
variability with a view to hybrid breeding, but also to broaden the genetic base
in B. napus with respect to pest and disease resistances. For such interspecific
hybridizations a variety of biotechnological tools, for example embryo rescue
techniques or protoplast fusion, are used to circumvent incompatibility bar-
riers. In some cases RS rapeseed forms have resulted in successful release of
cultivars carrying novel resistance genes from the diploid species. For example,
Diederichsen and Sacristan (1996) successfully used protoplast fusion to trans-
fer resistance to clubroot (Plasmodiophora brassicae) from B. oleracea to
B. napus. Through advanced backcrossing a race-specific resistance was subse-
quently transferred from RS rapeseed progeny to elite winter oilseed rape, and
the winter oilseed rape varieties ‘Mendel’ (Table 4.1) and ‘Tosca’ derived from
this material were released in the early 2000s to specifically combat this disease
in affected areas of Great Britain and Germany. In another example, Mithen
and Magrath (1992) generated synthetic lines of B. napus carrying resistance to
blackleg disease (Leptosphaeria maculans, anamorph: Phoma lingam) derived
from B. rapa via embryo culture. The resistance was then integrated successfully
into spring canola, resulting in the release of the cv. ‘Surpass’ in the late 1990s
and subsequent efforts to introgress this resistance into winter oilseed rape
material. This Phoma resistance from B. rapa has in the meantime been over-
come by virulent L. maculans isolates in Australia, and the clubroot resistance
from B. oleracea is also race-specific and hence not durable without careful
agronomic management (e.g. crop rotation). Nevertheless, these examples
demonstrate the potential utility of B. oleracea and B. rapa for the identification
and combination of novel resistance genes to important oilseed rape pathogens.
Table 4.1 Breeding oilseed rape cv. ‘Mendel’ resistant to clubroot (Plasmodiophora brassicae)
through introgression of clubroot resistance from Brassica rapa
Breeding stage Breeding material/activity
Sources Resynthetic rapeseed ‘1543’ (ECD-04 ECD-15)
ECD-04 ¼ B. rapa ssp. rapifera (resistent)
ECD-15 ¼ B. oleracea var. acephala cv. Verheul
Backcross to modern oilseed rape (Falcon ‘1543’) Falcon
Production of DH-lines 3,437 lines (NPZ-lab þ SU-lab)
Selection for resistance, 00-quality, and seed yield
Male parent Selection of Bl. 6431/96 as male parent
New F1 hybrids with females MSL004C and MSL007C, e.g. cv. ‘Mendel’
This strategy has the potential to prove particularly valuable for develop-
ment of resistance to Verticillium wilt. This disease, caused by the host-adapted
pathogen V. longisporum, causes serious yield losses in affected areas of Sweden,
Denmark, Great Britain and the north of Germany. The fungus forms micro-
sclerotia which can persist in the soil for more than a decade, and because
4 Oilseed Rape 97
accredited fungicides are not available the only current alternative for effec-
tive control of the disease in short crop rotations is the breeding of resistant
cultivars. Very little resistance is available in either winter or spring rapeseed,
thus necessitating a search for resistance sources in related species. Transfer of
resistance from B. oleracea to B. napus was reported by Happstadius et al.
(2003), while Rygulla et al. (2007a,b) reported the combination of resistances
from B. oleracea and B. rapa in novel RS B. napus genotypes by interspecific
hybridization, assisted by embryo rescue. After characterizing the resistance
by genetic mapping (Rygulla et al. 2008) it should be possible using marker-
assisted backcrossing to simultaneously transfer A- and C-genome resistance
genes into elite rapeseed lines, as a starting point for the development of new
cultivars with combined resistance from the diploid progenitors.
Interspecific crosses are also an important source of seed colour variants for
breeding of light-seeded B. napus. Brown or yellow seeds are of particular interest
for breeding of oilseed rape because of their association with a thinner seed coat
resulting in reduced dietary fibre content. This considerably improves the feed
quality of rapeseed meal after oil extraction (Shirzagedan and Röbbelen 1985;
Slominski et al. 1994, 1999). Light seed colour and low fibre content are con-
sidered to coincide because the biochemical pathways leading to lignin (fibre)
and pigment synthesis have common precursors such as pcoumarate (Theander
et al. 1977; Whetten et al. 1998). Furthermore, the reduction in testa thickness in
yellow-seeded oilseed rape has also been found to be associated with increased
seed oil and/or protein content per dry weight (Xiao and Liu 1982; Piotrowska
et al. 2003). A variety of different yellow-seeded rapeseed materials has been
generated by interspecific crosses between yellow-seeded B. rapa and brown-
seeded B. oleracea (Schwetka 1981) or B. alboglabra (Chen et al. 1988; Rahman
2001, 2003). The yellow-seed trait has also been introduced to B. napus from
B. chinensis (Liu 1983), B. juncea (Rashid et al. 1994) and B. carinata (Rashid
et al. 1994; Meng et al. 1998; Rahman 2001, 2003).
Other Brassica species and even less closely-related genera are also impor-
tant as potential sources of disease resistance for oilseed rape breeding. A
prime example for this is the use of interspecific and intergeneric hybrids as a
source for new resistance against blackleg disease. The genetic basis of black-
leg resistance in B. napus in European cultivars originates for the most part
from the French cultivar ‘Jet Neuf’, characterized by a polygenically con-
trolled adult plant resistance not expressed at the seedling stage (Cargeeg and
Thurling 1980). In contrast, all Brassica species containing the B genome
exhibit an absolute and stable resistance to most of the aggressive pathogen
isolates studied to date; B genome resistance is mono- or oligo-genically
controlled (see Rimmer and van den Berg 1992; Dixelius 1999) and efficient
from the seedling stage onwards. Thus, B genome donors like B. nigra (L.)
Koch (BB, 2n ¼ 16) and B. juncea (L.) Czern (AABB, 2n ¼ 36) have been
extensively used as genetic pool in an attempt to develop resistant oilseed
rape (e.g. Roy 1978; Sacristán and Gerdemann 1986; Sjödin and Glimelius
1989; Chèvre et al. 1996; Plieske et al. 1998; Dixelius 1999). On the other hand,
some aggressive isolates of the pathogen have been shown to overcome the
resistance of B. juncea (Purwantara et al. 1998). Leptosphaeria maculans
exhibits a broad variation in virulence, giving it the potential to adapt quickly
to a given resistance (Kuswinanti et al. 1999). Generation of durable resis-
tance therefore necessitates the application of a broad spectrum of resistance
sources in oilseed rape breeding. For this reason, interspecific and intergeneric
transfer of blackleg resistance from wild crucifers is an interesting alternative,
and in recent years progress has been made to introgress resistance into oilseed
rape from different sources, including Sinapis arvensis (Snowdon et al. 2000;
Winter et al. 2003) and Coincya monensis (Winter et al. 2003). Other examples
of intergeneric hybridisation for resistance gene transfer into B. napus include
resistance to beet cyst nematodes on Raphanus sativus addition chromosomes
(Thierfelder and Friedt 1995; Voss et al. 2000), whereas Klewer et al. (2003)
used sexual and somatic hybridisation in an attempt to transfer resistance to
Alternaria blackspot into B. napus from B. elongata, Sinapis alba, Diplotaxis
tenuifolia and D. erucoides. In such broad intergeneric hybrids ovary culture
techniques are absolutely necessary to overcome incompatibility barriers,
however a successful transfer of the desired trait is sometimes achieved. The
prerequisite for this is that intergenomic chromosome recombination takes
place in early backcross generations before the loss of non-homologous donor
chromosomes.
4.5 Major Breeding Achievements
Oilseed rape has become a major international crop only over the course of the
past three decades. This rapid advance to one of the major arable crops is a result
of spectacular breeding success. The oil from rapeseed and most other brassicas
naturally contains a high quantity of erucic acid (C22:1, cis 13-docosenoic acid),
which has a bitter taste and in high doses has been implicated in cardiac health
problems. This serious limitation to rapeseed as a foodstuff was overcome only
by the development of ‘0’ and ‘00’ rapeseed varieties in the 1970s (Stefansson
1983; Downey and Röbbelen 1989; Downey 1990). The first major breakthrough
came with the initial 0-quality cultivars with erucic acid levels of less than 1%
(Stefansson and Hougen 1964). Earlier rapeseed cultivars contained up to 50%
erucic acid in the seed oil (Table 4.2). The identification of the fatty acid mutants
from which the first 0-rapeseed derived was made possible by major improve-
ments in high-throughput seed analysis techniques, in particular gas chromato-
graphy. The first erucic acid-free variety, derived from a spontaneous mutant of
the German spring rapeseed cultivar ‘Liho’, was released in Canada in the early
1970s. The value of the crop was still suppressed by the presence of high quan-
tities of glucosinolates in the seed, however, which made rapeseed meal unsui-
table as a livestock feed. In monogastric animals the digestion of glucosinolates
results in the release of toxic by-products that can cause liver and kidney damage
4 Oilseed Rape
Table 4.2 Variation of seed lipid composition in different types of oilseed rape (B. napus)
Fatty acid1 composition (%)
Oil type Breeding method 12:0 14:0 16:0 18:0 18:1 18:2 18:3 20:1 22:1 others
Canola (00-quality rapeseed oil) Mutation and breeding – – 4 2 60 21 10 – – 3
High lauric Genetic engineering 37 4 3 1 33 12 7 – – 3
High myristic Genetic engineering – 18 23 2 34 15 4 – – 4
High stearic Genetic engineering – – 4 29 15 19 22 1 – 10
High oleic (HO) Mutagenesis – – 4 2 80 5 5 2 – 2
High oleic (HO) Genetic engineering – – 4 1 84 5 3 1 – 1
Low linolenic Mutagenesis – – 4 2 61 28 3 1 – 1
Low linolenic Genetic engineering – – 4 2 68 22 1 1 – 2
1
Major fatty acids: C12:0 lauric, C14:0 myristic, C16:0 palmitic, C18:0 stearic, C18:1 oleic, C18:2 linoleic, C18:3 linolenic, C20:1 eicosenic, C22:1 erucic
acid
99
along with lymph dysfunction. In 1969 the Polish spring rape variety ‘Bronowski’
was identified as a low-glucosinolate form, and this cultivar provided the basis for
an international backcrossing program to introduce this polygenic trait
(‘Bronowski’ was found to possess at least three recessive genes for low glucosi-
nolate content) into high-yielding erucic acid-free material. The result was the
release in 1974 of the first 00-quality spring rapeseed variety, ‘Tower’, with zero
erucic acid and low glucosinolate content, and thus marking the begin of the
advance of oilseed rape (canola) to one of the most important oil crops in
temperate regions in the following decades.
Another important achievement regarding seed oil quality is the development
of high oleic acid, low linolenic acid (so-called HOLL or HOLLi) types. The oil of
varieties such as ‘Splendor’ or ‘Nexera’ is characterized by oleic acid contents of
more than 75% and linolenic acid contents of less than 3% (Table 4.2). This gives
the oil a substantially higher oxidative stability, which is particularly beneficial
when used as a frying oil because the formation of deleterious trans-fatty acids is
strongly reduced. These new varieties are the result of experimental mutagensis
and conventional selection, where at least three major genes had to be modified to
achieve the HOLLi phenotype. Therefore, it is not surprising that this phenotype
is associated with a yield penality due to linkage drag. However, intensive breed-
ing activities using molecular breeding tools are expected to provide new high
yielding HOLLi oilseed rape cultivars in the near future.
With regard to enhancing the seed yield potential of rapeseed, the develop-
ment of functional male sterility systems for the production of true hybrid seed
has definitely been an enormous achievement. Present-day rapeseed hybrids are
single crosses based on two parental inbreds. For developing male sterile
females, both systems of cytoplasmic male sterility (e.g. the Ogura CMS intro-
duced from radish) and genic male sterility (GMS, controlled by nuclear genes
only) are commercially used, particularly in Europe. The first winter oilseed
rape hybrid varieties were registered in 1995 (Frauen and Paulmann 1999) and
are now covering a large part of commercial (winter) rapeseed production in
Europe.
The Male Sterility Lembke (MSL) GMS system is based on a spontanous
mutant selected in the nursery of the German breeding company Norddeutsche
Pflanzenzucht HG Lembke in the early 1980s. The MSL system allows the
production of fully restored rapeseed hybrids without any yield or quality
penalty, and all B. napus genotypes function as restorers. The INRA-Ogura
CMS system from radish (Ogura 1968), introduced to B. napus by INRA,
France, relies on an introgression from the radish genome including the Rf
gene for fertility restoration. By recombination and pedigree breeding, Pioneer
Hi-Bred scientists identified an Rf line with very low and stable glucosinolate
content, allowing the production of fully restored hybrids with canola quality.
In more recent lines the length of the original radish introgression sequence is
significantly reduced, leading to improved agronomic and quality characters
(Delourme et al. 1998).
4 Oilseed Rape 101
The general goals of oilseed rape breeding are summarized in Table 4.3. Major
goals with high or very high priority are: tolerance to late planting and winter
hardiness, plant height and lodging resistance, resistance to blackleg disease,
Verticillium wilt and (if possible) sclerotinia, very low contents of erucic acid
and glucosinolates, high oil content and marketable seed yield (M. Frauen and
others, personal communication).
Table 4.3 Four major fields and associated detail traits of oilseed rape breeding
Agronomic traits Disease and pest resistance
– Tolerance to late planting – Phoma and Vericillium
– Winter hardiness – Clubroot and Cylindorsporium
– Plant height and lodging resistance – Sclerotinia (resistance to be identified)
– Ripening time (early maturity) – Virus resistance (TuYV)
– Nutrient efficiency and drought – Various insects pests (pant resistances remain
tolerance to be identified)
– Shattering resistance
– Herbicide tolerance
Yield potential Seed quality
– Oil content – Very low erucic acid content (C22:1 <0.2%)
– Seed yield components – Low glucosinolate content (<18 mmol/kg seed)
– Harvest index – Reduced fibre (lignin) content and improved
– Total and marketable seed yield digestibility (monogastric animals)
Source: NPZ-Lembke, see Christen and Friedt (2007).
4.6.1 Seed and Oil Yield Potential and Stability

After the widespread adoption of 00-quality as the accepted standard for the
use of the crop in human and animal nutrition, the major focus in breeding efforts
returned to improving the seed and oil yield along with yield stability (Fig. 4.2).
With respect to morphological and agronomical characteristics, the yield of oil-
seed rape is composed of the number of siliques per unit area, the number of seeds
per silique and the 1000-seed weight (Diepenbrock 2000). Improvement of pro-
ductivity encompasses several agronomic parameters such as early maturity,
resistance to lodging and shattering as well as resistance to weeds, insects and
particularly to the major diseases.
The most significant diseases of oilseed rape in most major growing regions
are sclerotinia stem rot (Sclerotinia sclerotiorum) and stem canker (Lepto-
sphaeria maculans, anamorph: Phoma lingam), also known as blackleg disease
(Fig. 4.3); however, there are also a number of additional diseases of local
importance (Table 4.4). Verticillium wilt caused by Verticillium longisporum is
a particular problem in Sweden and Germany, light leaf spot (Pyrenopeziza
brassicae) in northern parts of Europe and clubroot (Plasmodiophora brassicae)
in Scandinavian countries and the northern United Kingdom.
Fig. 4.2 Rapeseed performance trials for identification of candidate varieties at a commercial
breeding station (photo: U. Baer, NPZ-Lembke, Germany)
Fig. 4.3 Incidence of phoma leaf spots (blackleg disease) caused by Leptosphaeria maculans
(anamorph: Phoma lingam) on a young leaf of oilseed rape in autumn (photo: U. Baer, NPZ-
Lembke, Germany)
4 Oilseed Rape 103
Table 4.4 Agronomical relevance, potential damage, possibility of control by chemicals or

genetic resistance, and priorities for further research regarding major pathogens and pests of
oilseed rape
Potential Chemical Genetic Need
Agronomical risk of prophylactic resistance for
Pathogen or pest relevance damage control available research
TuYV (virus) þþþ þþ – þ þ
Plasmodiophora þ þ þþ þ monogenic þ/–
brassicae
Peronospora þ þ/– þþ ? –
parasitica
Leptosphaeria þþþ þþþ þþ þ þþ
maculans
Sclerotinia þþþ þþþ þþ (?) þþþ
sclerotiorum GMO
Verticillium þþþ þþ – þ þþ
longisporum
Alternaria (þ) þ/– (þ) þ –
brassicae
Cylindrosporium – – (þ) ? þ
concentricum
Fusarium þ/– þ – þ –
oxysporum
Nematodes ? ? – þ (?) ?
Insect pests þþþ þþþ þþ resistance (?) GMO þþ
Breeding for increased disease resistance in oilseed rape involves a range of

strategies. For a number of diseases, including light leaf spot and blackleg disease,
a broad degree of natural variation is present in the species and has been
successfully used to breed varieties with more or less sustainable, quantitative
resistance. For other diseases, particularly sclerotinia stem rot, Verticillium wilt
and clubroot disease, very little resistance or tolerance is present in modern
varieties. Cultural control methods, particularly rotation, are therefore particu-
larly important for control of clubroot and sclerotinia, however in the case of
sclerotinia the use of chemical plant protection is still vital for efficient disease
control. For clubroot and Verticillium, on the other hand, effective fungicides are
not available. Due to the long persistence of these fungi in the soil the use of
resistant varieties is therefore the only way for effective long-term control of these
diseases in affected regions where the production of oilseed rape in crop rotations
is steadily increasing. In some cases transfer of resistant germplasm to B. napus
from other Brassica species and related crucifers has been successfully applied.
4.6.2 Improvement of Seed Components

Seeds of oilseed rape contain both valuable (nutritional) and anti-nutritional
compounds (Table 4.5). The gross seed composition can vary widely depending
Table 4.5 Major rapeseed compounds determining the feeding value for farm animals
Compounds determining feed value Antinutritive or undesired compounds
Seed oil (45%): Fatty acids Glucosinolates (isothiocyanates)
Sinapine (‘‘stinking eggs’’ of brown laying hens)
Storage protein (23%): Amino acids Lignin (restricting energy content)
Phytic acid (availability of P by animals)
Source: K.H. Südekum, personal communication.
on both genetic and environmental factors, with a large influence of tempera-

ture, water and nutrient supply. The oil content ranges from around 36 to 50%
(on a dry matter basis), while the oil-free meal contains 33–48% protein
(Canvin 1965; Appelqvist and Ohlson 1972; Arnholdt and Schuster 1981;
Marquard and Schuster 1981; Salunkhe et al. 1992). The improvement of oil
content is an important breeding goal due to the primary economic value of the
oil component and its relatively high heritability (Grami et al. 1977). Increasing
oil content has to date been quite successful due to the ease and speed with
which oil content can be measured by non-destructive NMR (nuclear magnetic
resonance) techniques. Although oil and protein content are negatively corre-
lated, improvements can be achieved through selecting for the sum of the two
seed components (Grami et al. 1977; Arnholdt and Schuster 1981; Stefansson
1983). However, low glucosinolate rapeseed meal still presents several problems
for use in livestock feeds. Besides the presence of undesirable compounds like
sinapic acid esters, phytic acid and phytates, phenolic acids and tannins, the
comparatively high crude fibre content (approx. 15% of dry oil-free meal) is
disadvantageous (Shahidi 1990; Thies 1991; Salunkhe et al. 1992). Due to the
small size of the seeds the hull, accounting for about 10–20% of the seed weight,
imparts most of the fibre content to the meal (Appelqvist and Ohlson 1972;
Anjou et al. 1977). Currently, the most promising route to reducing fibre and
hull content genetically, is to breed cultivars with a yellow (light) seed coat, like
the pure yellow-seeded cultivars occurring in the sarson subspecies of B. rapa or
in B. juncea. Since yellow seed coats are significantly thinner than brown or
black ones, the development of pure yellow-seeded B. napus cultivars with
agronomically acceptable performance still remains an important goal in qual-
ity breeding towards increased oil and protein content.
The value and suitability of rapeseed oil for nutritional or industrial pur-
poses is again determined by its fatty acid composition. The identification of
naturally occurring zero-erucic mutants in both B. napus and B. rapa was indeed
the first discovery opening the era of mutant-derived quality improvement in oil
crops (Downey 1964; Stefansson and Hougen 1964; Röbbelen 1990). Canola-
quality rapeseed low in saturated fatty acids and almost lacking nutritionally
undesirable very long-chain fatty acids meets all the requirements of a prime
edible oil (Ackman 1990; Downey and Bell 1990; Trautwein 1997). Despite the
beneficial nutritional properties of a-linolenic acid (18:3n–3), the oxidative sta-
bility of the oil can be improved by decreasing the linolenate content from an
4 Oilseed Rape 105
average of 10% to less than 3%, which results in enhanced shelf life (Rakow
et al. 1987; Pleines and Friedt 1988; 1989; Downey and Bell 1990) and a
reduction of trans-fatty acids. The latter is a particularly important nutritional
quality characteristic for high-temperature frying oils in the fast-food and food-
processing industries (Mensink and Katan 1993). In the last three decades
improvements of the C18 fatty acid composition in rapeseed (B. napus) were
achieved by selecting altered linoleate/linolenate genotypes after chemical
mutagenesis. Initially, the fatty acid profiles of these lines indicated that nearly
all of the linolenic acid was being directed to linoleic acid and that the level of
oleate increased only insignificantly (Rakow 1973; Röbbelen and Nitsch 1975;
Röbbelen and Thies 1980; Rakow et al. 1987; Röbbelen 1990). In 1988, the
spring rapeseed cultivar ‘Stellar’, which produces oil containing less than 3%
linolenate, was released for commercial production in Canada, although its
agronomic performance was less than satisfactory (Scarth et al. 1988). Today
HOLL or HOLLi varieties provide an important new quality of rapeseed oil for
nutritional purposes (Table 4.2).
Due to strong environmental and marked maternal influences, only low
correlations have been found between the contents of polyenoic fatty acids
determined in half-seeds and their progenies. The most important factor influ-
encing the biogenesis of the unsaturated fatty acids is the prevailing tempera-
ture during seed development (Pleines and Friedt 1988, 1989). Recent reports
described mutants with reduced levels of polyunsaturated fatty acids (PUFAs)
obtained by blocking oleic acid desaturation. The development of canola
cultivars with reduced levels of PUFAs accompanied by higher oleate content
would produce a dietary oil with additional markets (Marsic et al. 1992). For
industrial applications a very high content of oleic acid (80–90%) is preferred
because this is most suitable for consecutive chemical synthesis reactions (Lühs
and Friedt 1994).
As a facultatively outcrossing but predominantely self-pollinating species, oil-

seed rape is traditionally bred by classical line-breeding methods, and the
majority of oilseed rape cultivars are pure lines derived from breeding schemes
designed for self-fertilizing crops, i.e. pedigree selection or modifications
thereof. However, since the discovery and development of male sterility systems
in B. napus hybrid breeding has become an equally important aspect of cultivar
development in oilseed rape. Due to the generally high response of B. napus
genotypes to microspore culture techniques, the use of doubled haploid (DH)
production (see Section 4.8.1) has also become common practice in commercial
breeding programs and has already resulted in numerous licensed cultivars.
Doubled haploids are today also widely used for production of homozygous
parental lines for breeding of oilseed rape hybrids.
4.7.1 Traditional Line Breeding
By selection against self-incompatibility alleles B. napus can be developed as a

predominantly self-pollinating species. In the past, corresponding breeding
schemes have been adopted for developing open pollinated (OP) rapeseed
varieties with a moderate degree of inbreeding, also called ‘line varieties’. In
this case breeding is a rather straight-forward process including repeated plant
selection (first for highly heritable traits) starting in segregating F2 populations
from cross combinations of suitable parents (‘‘combination breeding’’). After
achieving adequate homogeneity, field observations (e.g. diseases, agronomic
traits) and subsequently replicated yield tests with continuously increasing
complexity (locations, years) are carried out. This leads to the identification
of suitable variety candidates to be submitted to official tests by the respective
plant variety office, e.g. the European Community Plant Variety Office (CPVO,
Angers/France) or the German Bundessortenamt (Hannover/Germany).
Repeated tests are the basis for variety release and are followed by more
practice-oriented yield tests before marketing of recommended varieties can
commence (Table 4.6).
This classical scheme was modified and improved by the development of
efficient haploid techniques for oilseed rape. Today, most breeding companies
will derive microspore cultures from F1 plants and regenerate doubled haploid
(DH) plants thereof. Since DH lines are completely homozygous, the subse-
quent breeding procedure can be accelerated and run more efficiently: First
field trials for yield estimation can be done one year earlier than in the classical
scheme, and the whole testing period can be reduced (e.g. 2 instead of 3 years).
Therefore, variety candidates can be submitted earlier for registration and the
marketing of successful candidates can start a couple of years earlier than with
lines developed via pedigree breeding (Table 4.6). In addition, homozygous DH
lines are very suitable for use in hybrid breeding, depending on their per se
performance and combining ability.
4.7.2 Hybrid Breeding and Cytoplasmic Male Sterility Systems
Although for many years the emphasis in oilseed rape breeding was strongly
focused on open pollinating varieties, up to 30% heterosis for seed yield has
been reported for B. napus (e.g. Schuster 1969; Grant and Beversdorf 1985;
Lefort-Buson et al. 1987; Brandle and McVetty 1989), and for both winter
rapeseed and spring canola hybrid varieties have rapidly gained in importance
over the past decade as effective systems for controlled pollination were devel-
oped. In current European winter rapeseed material yield improvements of up
to 15% have been reported for F1 hybrids compared to non-hybrid open-
pollinated varieties. This has led to a major increase in production of hybrid
rapeseed in the leading producing countries. For example, although only 21
Table 4.6 Generalized scheme for breeding of OP (line) varieties of oilseed rape – classical pedigree selection (left) versus haploid method (right)
Pedigree Haploid
breeding method
Year generation Material Procedure step Material Procedure
1 F1 (S0) Cross progeny Propagation F1 Cross progeny Microspore culture
4 Oilseed Rape
(androgenesis)
2 F2 (S1) Segregating population Plant selection A1 Segregating DH plants Selection and
propagation
3 F3 (S2) Selfed plant progenies Field observations A2 DH lines Replicated field test of
progenies (1 location)
4 F4 (S3) Inbred lines Replicated field test of A3 DH lines Replicated yield test of
progenies (1 location) progenies (>2
locations)
5 F5 (S4) Inbred populations Replicated yield test of DH lines Testing of variety
progenies (2 locations) candidates (1)
6 Inbred populations Replicated yield test of DH lines Testing of variety
progenies (>2 candidates (2)
locations)
7 Inbred populations Testing of variety Certified seed Testing of variety
candidates (1) production, variety candidates (3)
registration
8 Basic seed production Testing of variety Official variety trials
candidates (2)
9 Certified seed Testing of variety Start of marketing Official variety trials
production, variety candidates (3)
registration
10 Official variety trials
11 Start of marketing Official variety trials
F1–F5 ¼ Filial generations; S1–S4 ¼ Selfing generations; A1–A3 ¼ Androgenetic generations.
107
(28%) of the 76 German winter rapeseed cultivars listed by the German Plant
Variety Office in 2008 were hybrids (Bundessortenamt 2008), more than 50% of
the 1.5 million hectares of German winter rape in 2007/2008 were planted with
hybrid varieties. Already in 2003/2004 the hybrid cultivar ‘Talent’ had replaced
the open-pollinating ‘Express’ as the most widely-cultivated winter oilseed rape
variety in Germany, the first time a hybrid cultivar had achieved the top
position. One of the most important reasons for the upsurge in interest in hybrid
varieties is that they tend to have higher yield stability and better adaptation to
low-input cropping systems than conventional cultivars (Budewig and Léon
2003; Friedt et al. 2003).
Numerous cytoplasmic male sterility (CMS) systems have been discovered
and are used in brassica crops. Because CMS arises from specific interactions
between the mitochondrial and nuclear genomes, the combination of cytoplasm
and nucleus from different species often results in complete or partial male
sterility and in many cases functional mutations of floral structure. Two spon-
taneous male sterile cytoplasms, nap and pol, are found in B. napus. The nap
system was the first to be identified, originating from intraspecific crosses using
‘Bronowski’ (Thompson 1972) or ‘Hokuriku 23’ (Shiga and Baba 1973) as the
male parent. Most other B. napus CMS systems also result from interspecific or
intergeneric crosses, often using known sterility-inducing systems from other
species. The best example for this is the widely used ‘INRA-Ogura’ CMS
originating from Raphanus sativus (Ogura 1968), which was transferred to
oilseed rape by French scientists some 30 years ago (Bannerot et al. 1974).
Although this system was described by Tokumasu (1951) as a genic male
sterility, in B. napus it is expressed as cytoplasmic male sterility. The ‘Ogura’
CMS in radish (Raphanus sativus) is caused by an aberrant mitochondrial gene,
orf138, that prevents the production of functional pollen without affecting
female fertility. Rfo, a nuclear gene from radish that restores male fertility,
alters the expression of orf138 at the post-transcriptional level.
Although numerous CMS systems are available from different sources, their
use in oilseed rape breeding is often inhibited by instability, the absence of
suitable restorer or maintainer lines, or negative effects of the cytoplasm used to
induce the male sterility. Environmental instability of the expression of nap
male sterility means this system is unsuitable for hybrid production, and the
‘Polima’ (pol) system was only made workable by screening of huge numbers of
lines in different environments (Bartkowiak-Broda et al. 1991) in order to
identify stable maintainer genotypes. The monogenically inherited restorer
genes for B. napus ‘Polima’ CMS can be readily introduced into elite lines,
and pol is therefore now effectively used to produce registered F1 hybrid spring
canola varieties in numerous countries. Male-sterile inducing cytoplasm can
also have negative effects on flower morphology, nectar production, or on yield,
and sometimes chlorophyll deficiencies also need to be overcome. In some cases
suitable B. napus restorer lines have been produced for B. tournefortii CMS
(Banga et al. 1995; Stiewe et al. 1995a,b). Restored F1 hybrids based on the
‘Ogura’ CMS system are under increasing production in France, Germany and
4 Oilseed Rape 109
Fig. 4.4 Male fertile (left) and genic male sterile (Male Sterility Lembke; right) rapeseed
flowers (photos: U. Baer, NPZ-Lembke, Germany)
other European countries. Hybrid cultivars based on the commercial ‘Male-

Sterility Lembke’ (MSL) system are also prominent among winter oilseed rape
varieties in Germany. The advantage of this genic male sterility system (Fig. 4.4)
is that all B. napus lines restore fertility.
4.8 Introduction of New Biotechnologies into Breeding Programs
4.8.1 Tissue Culture and Haploid Techniques
Brassica napus is one of the most amenable crop species to improvement

through biotechnology. For instance, it is possible to reproducibly obtain
haploid and subsequently doubled-haploid (DH) plants through anther and/
or microspore culture (e.g. Weber et al. 2005). The principle advantage of
haploidy techniques is the rapid fixation of segregating genotypes, occurring
in lower frequency, in which recessive genes coding for specific traits are
combined in the homozygous condition. Thus, utilization of microspore culture
can allow a substantial acceleration of the breeding cycle. Besides haploid
techniques, wide hybridizations using embryo rescue techniques or protoplast
fusion can also be used to create novel genetic variation. This has been a
particularly successful method for integration of new disease resistance genes
from related species into oilseed rape.
4.8.2 Genetic Modification
Oilseed rape is particularly amenable to Agrobacterium tumefaciens mediated

transformation, and during the last two decades considerable progress has
been achieved in the development of genetically modified (GM) varieties.
Consequently, the global area of transgenic crops has grown continuously.
Beside soybean, cotton and corn, canola is one of the four principal crops in
which GM technologies are utilized. Herbicide tolerant canola was grown
on 3.6 million hectares, equivalent to 5% of the global transgenic area, in
2003. A limited public acceptance and unclear administrative legislation in
European Union member states has prevented expansion of GM traits into
European oilseed rape varieties. In Canada, on the other hand, herbicide-
resistant GM canola currently (2008) comprises about 80% of the canola
production in western Canada (see http://www.canola-council.org/). The
first glufosinate-ammonium tolerant B. napus spring cv. ‘Innovator’ was
registered for production in Canada in 1995 (Oelck et al. 1995). Although
weed control in canola is possible with available herbicides, multiple treat-
ments with chemicals of different herbicide families are often required for
control of all weeds. Certain cruciferous weeds such as wild mustard (Sinapis
arvensis) and stinkweed (Thlaspi arvense), are difficult to control, and the use
of speciality herbicides for cruciferous weed control is sometimes required. In
addition, the multiple uses of herbicides increases production costs and the
chemical load on soils. The availability of several types of herbicide tolerant
plants allows for rotation of herbicides, minimising the risk of weeds
becoming resistant to any particular chemical. Several varieties of transgenic
herbicide-tolerant oilseed rape are grown and processed in the USA, Canada
and China.
Genetic modification of the fatty acid composition is also an option to
make rapeseed oil more competitive in various segments of the food and
industrial oil markets. One of the central objectives in this context is the
genetic modification of the seed storage oil by maximising the proportion of
specific or functional fatty acids in order to obtain tailor-made raw materials
suited for various industrial purposes (Friedt and Lühs 1998; Biermann et al.
2000). However, the quality of vegetable food products has increased in
relevance for human nutrition in recent decades with the advent of so called
‘functional foods’. With regard to specific properties of such nutritive sub-
stances, genetic engineering offers the possibility to adapt plant storage lipids
to meet specific nutritional, industrial and even therapeutic requirements
(Leckband et al. 2002; Friedt et al. 2004). Rapeseed oil is unique in having a
large spectrum of usability and positive properties for food and non-food
applications. Genetic engineering of plant lipid biosynthesis in rapeseed has
already led to commercialisation, with transgenic varieties expressing geneti-
cally modified fatty acid patterns being available since 1995 (cf. Friedt and
Lühs 1998).
4 Oilseed Rape 111
4.8.3 Genetic Mapping, Genome Analysis and Marker-Assisted

Selection
Molecular markers have been widely used to map agronomically important

genes in oilseed rape and frequently play an important role in breeding and
selection procedures. The complete sequencing of Arabidopsis thaliana (The
Arabidopsis Genome Initiative 2000) and the development of comparative
genetic and physical maps between B. napus and Arabidopsis (e.g. Parkin
et al. 2005) has enormous potential for application in gene identification and
breeding in oilseed rape. The genome of B. rapa is expected to be completely
sequenced within the near future (for progress see http://www.brassica.info),
and current technological developments in the field of ultra-fast DNA sequen-
cing are beginning to revolutionise the fields of polymorphism discovery,
genome analysis and molecular breeding. The number of expressed sequence
tag (EST) sequences available for B. napus has skyrocketed in the past few
years as sequencing costs have diminished, enabling DNA sequence mining to
become extremely useful for the identification and development of single
nucleotide polymorphisms (SNPs) in oilseed rape. In the near future it can
be expected that high-density B. napus SNP arrays will play an important role
in development of dense genetic maps for oilseed rape. Next-generation
sequencing technologies are set to rapidly accelerate SNP discovery, so that
ultra-high density SNP maps will probably become commonplace in the near
future. High-throughput SNP screening methods will also be a valuable
resource for whole-genome allele-trait association studies, which potentially
will play a major role in the identification of genes contributing to complex
traits.
4.8.3.1 Genetic Maps and QTL Analysis

The first genetic map for B. napus was developed by Landry et al. (1991) using
restriction fragment length polymorphism (RFLP) markers. In the subsequent
two decades a large number of B. napus genetic maps were generated worldwide
using different rapeseed crosses, and considerable efforts have been invested in
the localisation of genes and QTL controlling agronomically relevant traits.
The most extensive B. napus genetic map published to date was an ultra-dense
map of 13,551 sequence-related amplified polymorphism (SRAP) markers that
were assembled into an ultra-dense bin map by Sun et al. (2007). A marker
density of 8.45 SRAPs per cM was achieved, which according to the authors
could correspond to more than one marker per 100 kb. This demonstrates the
great potential of ultra-dense mapping for map-based gene cloning; the avail-
ability of ultra-dense maps based on sequence-annotated SNP marker techni-
ques derived from next-generation sequencing or EST-SNP arrays will in the
near future hugely expand the opportunities for rapid discovery of candidate
genes for both simple and complex traits.
A detailed summary of B. napus crosses, mapping populations, marker

systems, map details and the quantitative traits that were studied until 2006 is
given by Snowdon et al. (2006). In the following we will describe some of the
more recent genetic mapping studies, including QTL analysis of traits that had
previously been less intensively studied, along with some novel approaches for
identification of genetic markers and candidate genes closely linked to impor-
tant traits in oilseed rape. In some cases marker-assisted genome scans were
implemented to introgress novel genetic diversity into oilseed rape breeding
lines.
As the importance of hybrid cultivars has increased over the past decade,
there has been growing interest in identifying the mechanisms and potential
genomic loci responsible for the manifestation of heterosis in oilseed rape.
Different strategies have been used to analyse the quantitative genetics of
heterosis for yield and related traits with the assistance of genetic maps. For
example, Radoev et al. (2008) mapped QTL contributing to additive, dominant
and overdominant heterosis effects in a mapping population of 250 doubled
haploid lines that were tested in two-year, multi-location field trials along with a
corresponding set of test hybrids from each of the DH lines with a common
male-sterile tester parent. Heterosis levels of up to 30% for grain yield were used
to map QTL involved in heterosis for yield and related seed traits. A large
number of epistatic interactions were found to interact with dominance and
overdominance effects to control expression of heterosis.
Use of genome-wide marker screens can also be useful for the introduction of
novel genetic diversity for the exploitation of heterosis in hybrid breeding. For
example Li et al. (2006) described a marker-assisted approach to develop new
types of B. napus with introgressions of A genome chromosomes from B. rapa
and C genome chromosome segments from B. carinata. When crossed with
conventional B. napus these new types demonstrated elevated levels of so-called
‘intersubgenomic heterosis’ for seed yield and related traits. Basunanda et al.
(2007) utilised dense whole-genome marker scanning to identify DH lines in
which the genes for zero erucic acid along with QTL for low glucosinolate
content were introgressed from a 00-quality variety into a novel genetic back-
ground of semi-synthetic ++ quality rapeseed. Test hybrids generated using
these genetically diverse introgression lines as pollinators showed high mid-
parent heterosis for seed yield (Basunanda et al. 2007; Gehringer et al. 2007).
Dissection of yield and yield component traits is another important aspect
that has been analysed extensively in oilseed rape by QTL analysis. For exam-
ple, Chen et al. (2007) recently reported on the detection of numerous QTL for
yield and yield-related traits in DH and immortalised F2 populations and found
some QTL that contributed significantly to numerous yield-related traits and
could be interesting targets for yield improvement. As the technologies for
highly-dense genetic mapping improve and it becomes possible to more accu-
rately integrate and compare map and QTL data from different populations, it
will be of great interest to see whether important QTL related to yield co-
localise in different materials, and whether such QTL may interact with
4 Oilseed Rape 113
epistatic loci involved in yield heterosis. Meta-analyses with multiple mapping

populations and large, common marker sets will hopefully enable such deter-
minations in the future.
4.8.3.2 Male Sterility

Considerable advances have been made in recent years in mapping and marker
development for genes controlling genic and cytoplasmic male sterility systems in
oilseed rape. For example, Yi et al. (2006), Lei et al. (2007) and Huang et al.
(2007) described the fine-mapping of three recessive genic male-sterility genes
using amplified fragment length polymorphism (AFLP1: Keygene, Wageningen,
The Netherlands) and amplified consensus genetic markers (AGGM) in large
segregating populations, and the anchoring of the linked markers to previous
B. napus genetic maps. In each case flanking marker sequences covering a region
of well under 1 cM in B. napus were used to delineate syntenic chromosome
regions in Arabidopsis that may contain the orthologs to the respective sterility
genes. Hong et al. (2008), Xie et al. (2008) and Xiao (2008) described the devel-
opment of sequence-based markers with tight linkage to an epistatic genic male
sterility suppressor gene, while He et al. (2008) generated sequence-characterised
markers linked to a cytoplasmic male sterility fertility restoration gene. In each of
these cases AFLP markers and bulked-segregant analysis played an important
role in whole-genome marker saturation to identify sequences with very lose
linkage to the responsible genes. Sequence annotations to Arabidopsis and an
often well-conserved synteny can assist greatly to identify potential candidates in
corresponding chromosome regions, and bulked-segregant analyses have proved
a valuable method to fine-map and clone genes involved in male sterility and
fertility restoration. In a different approach based on differential gene expression,
Wu et al. (2007) used suppressive subtractive cDNA techniques and cDNA
microarray hybridisation to try to identify candidate genes for a dominant
genic male sterility in B. napus. A number of genes involved in male gametogen-
esis pathways were among the differentially expressed genes between fertile and
sterile near-isogenic lines.
4.8.3.3 Oil Content and Quality

Identification and utilization of important genes contributing to oil content is
one of the major aims of seed quality breeding in oilseed rape. Fu et al. (2007)
mapped loci with major contributions to oleic, linoleic and linolenic acid con-
tent, and developed markers tightly linked to the responsible fatty acid desatur-
ase genes which can be used in effective selection of HOLLi genotypes. A recent
publication compared oil content QTL in different mapping populations and
revealed that some major gene loci appear to influence this complex trait in
different genetic backgrounds. Delourme et al. (2006) localised oil content QTL
in two large, genetically divergent mapping populations and compared their
locations to previously mapped QTL from earlier studies. In some cases the
QTL were found to be consistently revealed across different genetic back-

grounds. In particular, a QTL on N3 was revealed in all the studies and other
QTL on N1, N8 and N13 were found in three out of five different studies. Other
QTL were located in homeologous genome regions, while some were specific to
a particular genetic background and potentially carry novel alleles. These
results show the potential for combination of favourable alleles at different
QTL to increase seed oil content. Furthermore, examples were given of how
Arabidopsis genomic data could be used to derive markers and identify candi-
date genes for oilseed rape QTL. The study was also a good example demon-
strating the added value of consolidated information from different segregating
populations in order to identify meta-QTL involved in a specific trait in differ-
ent genetic backgrounds.
4.8.3.4 Yellow Seed Character

Much interest has developed recently with respect to the breeding of yellow-
seeded oilseed rape and canola with improved seed meal quality. The yellow-
seed trait in B. napus is generally associated with a reduced seed coat thickness;
this leads to a reduced contribution of the seed coat to the seed meal after oil
extraction and a consequent lowering of anti-nutritive crude fibre and phenolic
compounds. Unfortunately seed colour itself is difficult to use as a morpholo-
gical marker for improved meal quality because the accumulation of seed coat
tannins is highly sensitive to temperature, light intensity and other abiotic
factors. Therefore there is a considerable effort to identify major genes con-
tributing to reduced seed coat in different yellow-seed materials and develop
markers for effective breeding of high-performing light-seeded varieties. Badani
et al. (2006) localised a major QTL with a large contribution to seed colour and
acid detergent fibre content in two different yellow-seeded winter rapeseed
sources. The gene was flanked by markers originating from B. napus chromo-
some N18, although later work showed that the chromosome segment contain-
ing the major QTL derived from chromosome N9 and the responsible gene may
be present on a non-reciprocal translocation. In Chinese oilseed rape with a
completely different genetic background as compared to the previous study, Fu
et al. (2007) also found a major dominant QTL that appeared to be localised on
chromosome N9. Liu et al. (2005) and Xiao et al. (2007) also developed closely
linked markers to a major gene for yellow seed colour in Chinese oilseed rape. It
will be of great interest to determine whether the same major genes are influen-
cing seed colour-related traits in these genetically diverse materials, and also to
use the markers and candidate genes to identify new allelic diversity for these
traits among other oilseed rape materials.
4.8.3.5 Resistance to Biotic and Abiotic Stress

Mapping and marker development for resistance genes to biotic stress factors
are important goals in oilseed rape breeding. In recent years continued progress
4 Oilseed Rape 115
has been made in the map-based cloning of genes contributing to resistance

against blackleg disease, the major disease of oilseed rape worldwide (see
Rimmer 2006). Mayerhofer et al. (2005) described the fine mapping of loci
involved in seedling resistance to blackleg in two different canola cultivars.
Both loci localised to the same position on B. napus chromosome N7, and a
collinear chromosome region could be identified in Arabidopsis. A complex
pattern of tandem duplications was identified in the B. napus genome region
containing these loci. Apparently, duplication and sequence divergence during
the polyploidisation events that led to the Brassica species may also have played
a major role in the evolution of resistance to major pathogens. Candidate genes
for blackleg resistance were also identified in Arabidopsis by Staal et al. (2006);
fine-mapping was performed in recombinant inbred lines to identify two genes
that were associated with resistance and contained typical resistance gene
sequence motifs. The contribution to resistance was confirmed by reverse
genetics.
For a number of other diseases of oilseed rape breeding efforts have been
hindered by a lack of resistance sources. In some cases this can be overcome by
introduction of resistance genes from exotic B. napus materials, resynthesised
rapeseed or other interspecific crosses, however the availability of useful
selection markers is a prerequisite for effective combination of quantitative
resistances in elite germplasm. For example, Rygulla et al. (2007a) identified
QTL-linked markers associated with resistance against Verticillium longis-
porum introduced from B. oleracea, while Werner et al. (2008) described
QTL involved in resistance against clubroot disease (Plasmodiophora brassi-
cae). Resistance to Sclerotinia sclerotiorum is a major breeding aim in most of
the major oilseed rape growing areas of the world, however little resistance has
yet been identified against this disease in B. napus. QTL analysis of a partial
resistance in Chinese rapeseed lines was characterised by Zhao and Meng
(2003), and gene expression profiles produced by resistant and susceptible
genotypes in response to S. sclerotiorum infection were analysed by Zhao et al.
(2007) using microarray analysis. Early response genes to pathogen inocula-
tion were integrated into the QTL map, leading to the identification of a
number of candidate genes for the defense reaction. Among the genes that
co-localised with interesting resistance QTL, some plant cell wall-related
proteins and WRKY transcription factors were identified as potential con-
tributors to defence against sclerotinia rot.
Abiotic stress resistance is also gaining increasing attention in oilseed rape
breeding, although the regulatory mechanisms involved in whole-plant reac-
tions to drought conditions, nutrient deprivation or cold stress can be extremely
complex. Considerable efforts have been made to investigate genes involved in
vernalisation requirement and flowering time in B. napus and related species,
and in recent years it has become clear that some of the major genes controlling
flowering traits may play an important global role in regulation of gene expres-
sion in general. In recent years the role of FLOWERING LOCUS C (FLC)
homologs and associated genes in the regulation of flowering time and related
traits, and their involvement in relevant QTL for these traits, has been con-
firmed in B. napus and its diploid progenitors (e.g. Pires et al. 2004; Kim et al.
2007; Lou et al. 2007; Okazaki et al. 2007; Razi et al. 2008).
4.8.3.6 Novel Genomic Tools

The astonishing current developments in next-generation sequencing technol-
ogies offer unprecedented opportunities for new genomics-based breeding and
selection strategies. In particular, as soon as the sequencing of the reference A
genome of B. rapa is completed it will be possible to resequence large portions or
the B. oleracea C genome and consequently the A and C genomes of B. napus.
Even without a reference sequence, the next-generation sequencing technolo-
gies enable large-scale comparative sequencing of BAC libraries from elite
breeding lines for a relatively low and continuously decreasing cost, so that
whole-genome selection in oilseed rape and other major crops is likely to
become a reality in the foreseeable future. This new sequence-based genomics
era will probably completely change the way that genetic mapping, genome
analysis and marker-assisted selection are performed in crop plants. Presum-
ably, high-throughput chip-based screening of large segregating progenies will
play a major role in breeding and selection. Such techniques will also be a
pivotal technology in the application of whole-genome association genetics
methods for the identification and utilisation of genes involved in important
complex traits. Already the use of genome-wide transcriptome analysis has
enabled the identification of potential global gene expression regulators that
could be useful for significant yield increases. Ultra-deep expression profiling
with the help of next-generation sequencing has the potential to take this kind of
gene discovery to a new level. Although oilseed rape genome research has long
profited from the close relationship to Arabidopsis, one of the most intensively
studied plant species, in the near future it is likely that a vast array of genomic
tools will also be available for brassica crops. One major limitation to genome-
assisted breeding is the current lack of associations between genomic data and
detailed, reliable phenotypic data for factors contributing to major traits.
Intensive phenotyping, including high-throughput physiological and metabo-
lite profiling, may be the most important key to understanding important
complex traits such as oil content, seed developmental characters, biotic and
abiotic stress tolerance and the manifestation of yield characters in oilseed rape.
Novel and high-throughput phenotpying technologies should therefore be an
important priority in coming years in order to facilitate the identification of
genomic and transcriptomic variation associated with economically important
characters.
4.8.3.7 Utilization of Synteny to Arabidopsis

Synteny to Arabidopsis and increasing quantities of aligned genomic sequence
data from Brassica species are also helpful to identify candidate genes and
4 Oilseed Rape 117
potentially gene-linked markers for important traits in oilseed rape. To

demonstrate the power of synteny-based marker development, Hasan et al.
(2008) developed potentially gene-linked markers for seed glucosinolate loci
via structure-based allele-trait association studies in genetically diverse
B. napus genotypes. Association analyses were performed in a core set of
gene bank accessions, and included simple-sequence repeat (SSR) markers
whose orthologs in A. thaliana were expected to be physically closely linked to
promising candidate genes for glucosinolate biosynthesis. Using this
approach, four genes involved in the biosynthesis of indole, aliphatic and
aromatic glucosinolates were tested for associations to total seed glucosino-
late content in B. napus. Markers linked to homoeologous loci of all four genes
in B. napus were found to be associated with a significant effect on the seed
glucosinolate content. This example shows the potential of Arabidopsis-
Brassica comparative genome analysis for synteny-based identification of
gene-linked SSR markers that may be used in marker-assisted selection for
important traits in oilseed rape.
4.9 Seed Production

The appropriate procedure of seed production depends on the variety type, i.e.
open-pollinated (OP) cultivars versus hybrid cultivars. Since the former repre-
sent essentially homozygous inbred lines, they can easily be reproduced and
increased by cultivation in isolation, i.e. with sufficient distance to other oilseed
rape crops in order to exclude cross pollination.
In comparison, the procedure of hybrid seed production is much more
complicated. Current rapeseed hybrid cultivars are single cross F1 hybrids
based on two parental (female and male) inbred lines. For developing male
sterile females, both systems of cytoplasmic male sterility and genic male
sterility (controlled by nuclear genes only) are commercially used (see above).
Initially, the parental inbreds have to be propagated under strict isolation. In
the next step, F1 seed production is carried out in alternate stripe cultivation of
female seed parent and male pollinator lines (Fig. 4.5). For hybrid seed produc-
tion, attention has to be paid to specific requirements: minimum male sterility
of female lines must be 98% and the minimum hybridity of hybrid seed
harvested must be 90%. Hybridity is determined by counting typical male fertile
(restored) plants in field-grown seed progeny.
General requirements for seed characteristics and production include:
(1) Planting requirements for seed production (determined via field inspec-
tions), (2) seed characters including purity, germination rate, and moisture
level, (3) contamination with seeds from all other species, and (4) phytosanitary
requirements (live insect pests or mites, contamination with sclerotia of Scler-
otinia). In general, the requirements are more stringent for certified seed than
for basic seed (Table 4.7).
Fig. 4.5 F1 hybrid seed

production through
alternate strip cultivation of
male sterile (seed parent)
and male fertile (pollinator
parent) rows at different
developmental stages:
before flowering (top), full
bloom (center), and after the
end of flowering with male
parent removed (bottom)
(photos: U. Baer, NPZ-
Lembke, Germany)
Table 4.7 Minimum requirements for the production and purity of basic and certified oilseed
rape seed according to the German federal seed trade regulations
Basic Certified
Requirements for seed characteristics and production seed seed
Plant requirements for seed production (determined via field
inspections)
No. of non-typical rapeseed plants (off-types) which may cause 5 10
cross pollination (max. number of plants per 150 m2)
Species whose seeds are difficult to eliminate by cleaning (max. 10 15
number of plants per 150 m2)
Minimum distance (m) to other species whose pollen can cause 200 100
cross-fertilization
4 Oilseed Rape 119
Table 4.7 (continued)

Basic Certified
Requirements for seed characteristics and production seed seed
Seed characters
Purity (%) 98 98
Germination rate (%) 85 85
Residual moisture (%) 9 9
Contamination levels
Maximum contamination with seeds from all other species (% total 0.3 0.3
weight)
Wild mustard/charlock (maximum number of seeds) 10 10
Dock/sorrel, except common and golden dock (maximum number 2 5
of seeds)
Wild oats, oatgrass (maximum number of seeds) 0 0
Phytosanitary requirements
Live insect pests or mites 0 0
Sclerotinia sclerotiorum (number of intact or partial sclerotia) 10 10
Source: Adapted from Rutz (2004).
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Chapter 5
Other Brassicas
Leonardo Velasco and José M. Fernández-Martı́nez
5.1 Introduction
The genus Brassica is one of the most important genera contributing to agri-
culture. It includes a total of 41 species, six of them of economic importance:
B. juncea (L.) Czern., B. napus L., B. nigra (L.) Koch, B. oleracea L., and B.
rapa L., worldwide distributed, and B. carinata A. Braun, restricted to Ethiopia
and surrounding countries. Other species have been cultivated in the past, are
nowadays cultivated on a small scale, and/or wild types are locally used (Gladis
1989). The genus encompasses very diverse types of plants, grown as vegetables,
fodder, and sources of oils and condiments (Prakash and Hinata 1980).
Vegetable oil from Brassica spp. represents nowadays the third source of
vegetable oil in the world market. Most of the oil is obtained from double-zero
or canola cultivars, which produce seed oils with less than 2% erucic acid and
seed meals with less than 30 mmol of aliphatic glucosinolates per gram of oil free
meal (Downey and Rakow 1987). Double-zero germplasm has been developed
for B. napus (Downey et al. 1969), B. rapa (Kondra and Stefansson 1970), and
B. juncea (Love et al. 1990), whereas the development of double-zero germ-
plasm of B. carinata is underway (Márquez-Lema et al. 2006).
Brassica napus is the major oilseed crop of the genus and it is cultivated
worldwide. Other Brassica oilseed crops are cultivated at smaller scale,
although some of them are locally important (e.g. B. juncea and B. rapa in
India and Canada and B. carinata in Ethiopia) and in some cases their cultiva-
tion is spreading in recent years. Winter forms of B. rapa are mainly grown in
areas with severe cold climates such as parts of Sweden and Finland. The spring
cultivars of B. rapa are extensively cultivated in western Canada, parts of
Sweden and Finland, northwest China, and the Indian subcontinent. The latter
is also the major area of cultivation of B. juncea, although the crop is becoming
relevant in other areas such as Canada and Australia due to the recent avail-
ability of double-zero cultivars. B. carinata cultivation is still restricted to
L. Velasco (*)
Institute for Sustainable Agriculture (CSIC), Córdoba, Spain
e-mail: ia2veval@uco.es

128 L. Velasco and J.M. Fernández-Martı́nez
Ethiopia and surrounding countries. The following discussion deals with oil-
seed brassicas other than B. napus, which is reviewed in another chapter of this
volume.
Crop brassicas comprise three elementary species having homeologous gen-

omes named A (B. rapa; n=10), B (B. nigra; n=8), and C (B. oleracea; n=9),
and three amphidiploids that originated from natural hybridizations between
two of the elementary species: B. carinata (BC; n=17), B. juncea (AB; n=18),
and B. napus (AC; n=19) (U 1935). They have been cultivated since ancient
times as vegetables, fodder, and sources of oils and condiments (Prakash and
Hinata 1980). Current oilseed types mainly belong to B. napus, B. rapa,
B. juncea, and B. carinata. Brassica crops include very distinct forms that are
cultivated for different uses. Storage organs (turnips), leaves, and seeds are
utilized in the varying forms of B. napus, B. rapa, and B. juncea. B. carinata is
used in the Ethiopian plateau as a vegetable and as a source of oil. The first
records on the use of the seeds as spices date back to Sanskrit literature around
3000 BC (Prakash and Hinata 1980).
5.2.1 Brassica rapa
The species includes a vast number of morphologically divergent forms that are
cultivated as green vegetables or for their storage organs (turnips) and seed oils.
Two main centres of origin have been suggested: the Mediterranean area, which
is considered as the primary centre of origin of European forms, and eastern
Afghanistan and the adjacent region of Pakistan (McNaughton 1979). The
species is divided into seven subspecies, some of them considered for a long
time as separated species. Oilseed forms are included into subsp. oleifera (oil-
seed turnip or turnip rape), subsp. trilocularis (yellow sarson), and subsp.
dichotoma (brown sarson, toria) (Hanelt 1986). The subsp. oleifera has been
suggested to be the basic cultivated form nearest to the wild type (McNaughton
1979).
5.2.2 Brassica juncea
Brassica juncea is a mustard species, known as Indian or oriental mustard, with

great economic importance. It is used as an oil, vegetable, and condiment crop
(Vaughan and Hemingway 1959). It is generally accepted that B. juncea origi-
nated from B. nigra and B. rapa somewhere in the Middle East or Central Asia
(Prakash and Hinata 1980).
5 Other Brassicas 129
After B. oleracea, B. juncea is the species of the genus that exhibits the
greatest intraspecific variability, although it has been studied to a smaller extent
than B. oleracea (Hanelt 1986). The greatest diversity of forms occurs in India
and China. Cultivation has spread the species worldwide, especially to south-
ern- and south-eastern Asia (mainly Malaysia and Indonesia), the regions from
south-western Asia to northern Africa and south-eastern Europe, western
Europe (especially England), western Africa, and America (Hanelt 1986). The
species is divided into four subspecies, some of them mainly used as root
vegetables (subsp. napiformis) or as leaf vegetables (subsp. tsatsai, subsp. integ-
rifolia). The oilseed forms mainly belong to subsp. juncea (Gladis 1989).
5.2.3 Brassica carinata

Brassica carinata, commonly known as Ethiopian mustard, arose as a natural
cross between B. nigra and B. oleracea in north-eastern Africa, probably in the
Ethiopian plateau, where wild forms of B. nigra co-exist with cultivated forms
of B. oleracea since ancient times (Tsunoda 1980). The species is only found
under cultivation, mainly in Ethiopia and surrounding countries (Hanelt 1986).
The crop is used both as a leaf vegetable as well as an oil crop. Although some
authors have reported little differentiation into crop types (Labana and Gupta
1993), others have identified distinct leaf cabbage types of B. carinata (Tcacenco
et al. 1985). The species has been traditionally considered to contain low
intraspecific variability (Hanelt 1986), although recent morphological and
molecular studies have shown large genetic diversity within germplasm of this
species (Alemayehu and Becker 2002; Teklewold and Becker 2006; Warwick
et al. 2006).
5.3 Varietal Groups
5.3.1 Open Pollinated, Synthetic, and Hybrid Cultivars

Brassica rapa is an allogamous species with a high degree of self-incompatibility.
Conversely, the amphidiploid species B. juncea and B. carinata are partially
allogamous, with a high degree of self-pollination. Experiments in B. carinata
have shown an average outcrossing rate of 30% (A. Teklewold, personal com-
munication). Unlike B. napus, in which hybrid cultivars are replacing open
pollinated varieties, most cultivated varieties of the other oilseed brassicas are
open pollinated, mainly developed through pedigree selection (B. juncea,
B. carinata) or recurrent selection (B. rapa). Synthetic cultivars composed of
two or three parental lines are also used to exploit heterosis in B. rapa. Hybri-
dization systems based on both nuclear and cytoplasmic male sterility have been
developed for B. juncea (Pandey et al. 1999; Sodhi et al. 2006).
5.3.2 Winter and Spring Cultivars
Brassica rapa includes annual and biennial forms. The latter have vernalization
requirements to flower and they can be cultivated as winter crops only in areas
with cold winters. The former have practically no vernalization requirements
and they are cultivated as spring crops in areas with cold winters or as fall-sown
crops in areas with mild winters. Winter cultivars of B. rapa have been impor-
tant in the past, but nowadays they have been largely replaced in most areas by
more productive winter cultivars of B. napus. Only spring types exist of
B. juncea and B. carinata.
5.3.3 Wild-Type, Single-Zero, and Double-Zero Cultivars
The seed oil extracted from wild-type Brassica seeds contains high erucic acid
content, usually between 35 and 45% of the total seed oil fatty acids. Addition-
ally, the seed meal contains high glucosinolate content, commonly above
100 mmol g–1 seed. The toxic and antinutritive nature of both compounds has
represented a serious obstacle for the commercialization of seeds from wild-type
cultivars. The development of germplasm with seed oil free of erucic acid led to
the development of single-zero cultivars of B. rapa (Downey 1964), B. juncea
(Kirk and Oram 1981) and B. carinata (Alonso et al. 1991), whereas an addi-
tional drastic reduction of glucosinolate content produced double-zero culti-
vars of B. rapa (Downey et al. 1969; Jönsson et al. 1975) and B. juncea (Love
et al. 1990). Double-zero cultivars are also known as canola.
Brassica genetic resources are very rich in variability due to the large number of
closely related species. In addition to the three amphidiploid species (B. napus,
B. juncea, and B. carinata), which originated from spontaneous interspecific
hybridization between the diploid species (B. oleracea, B. rapa, and B. nigra),
there are other species of the genus Brassica and other related genera within the
tribe Brassiceae from which it is possible to identify useful variability that can
be transferred to turnip rape and mustard oilseed crops. A complete list of wild
germplasm of Brassica and allied crops has been published (Warwick et al.
2000). Germplasm resources are available for Brassica breeders through inter-
national bank collections. Important Brassica germplasm collections are main-
tained in Europe by the European Cooperative Programme for Plant Genetic
Resources (ECPGR) members. The database of these collections from 22 coun-
tries and 36 institutions include 19,600 accessions available in the ECPGR
database (http://www.cgn.wur.nl/pgr/collections/brasedb/). Other important
collections are maintained in the North Central Regional Plant Introduction
Station (NCRPIS) of USA (database available at http://www.ars-grin.gov/

npgs/searchgrin.html), the National Bureau of Plant Genetic Resources
(NBPGR) of India, and the Institute of Biodiversity Conservation and
Research (IBCR) of Ethiopia.
As described below in this chapter, these resources have been successfully
used in the identification and transfer of traits of interest, such as oil and meal
quality, disease resistance, and cytoplasmic male sterility to the oilseed species
B. rapa, B. juncea and B. carinata. However, due to the number and diversity of
germplasm collections it is difficult to select plant materials for specific objec-
tives. In order to provide efficient means of identifying useful traits, extensive
collections from European gene banks were documented, evaluated and char-
acterised within an EU research project (van Soest et al. 2004), and core
collections representing useful variability for Brassica oilseed breeding were
generated. These core collections constitute useful resources for identifying
valuable agronomic and quality traits that can be transferred to cultivated
brassicas. For example, the resulting core collection of B. rapa contains a
broad variation for disease resistance (Pinnegar et al. 2004). The evaluation of
the B. carinata material showed a wide variation for seed quality traits (Font
et al. 2004). Other germplasm evaluations in B. carinata collections identified a
high level of variability for agriculturally important traits (Alemayehu and
Becker 2002; Teklewold and Becker 2006; Warwick et al. 2006). Similarly,
evaluations of large germplasm collections of B. rapa and B. juncea have
identified a high level of variation for agronomic and quality traits in these
species (Knowles et al. 1981).

The most relevant breeding achievement in Brassica oilseed crops other than
B. napus have been related to the improvement of oil and meal quality, especially
the development of double-zero or canola cultivars of B. rapa and B. juncea.
5.5.1 Oil Quality
Germplasm free of erucic acid was first identified in B. rapa through single-seed
selection within the cultivar ‘Polish’ (Downey 1964). Zero-erucic acid germ-
plasm was developed in B. juncea through single seed selection in entries with
reduced levels of this fatty acid (Kirk and Oram 1981). In B. carinata, zero
erucic acid germplasm was developed through intraspecific selection (Alonso
et al. 1991) as well as through interspecific introgression of genes from B. juncea
(Getinet et al. 1994) or both B. juncea and B. napus (Fernández-Martı́nez et al.
2001).
Additionally to the elimination of erucic acid, seed oil quality of Brassica

oilseed crops has been improved through the development of germplasm with
low linolenic acid and/or high oleic acid content. Significant reductions of
linolenic acid levels have been achieved in B. rapa (Auld et al. 1992; Laakso
et al. 1999), B. juncea (Sivaraman et al. 2004), and B. carinata (Velasco et al.
2004). Mid and high oleic acid types have been also developed in B. rapa (Auld
et al. 1992; Tanhuanpää et al. 1996b), B. juncea (Stoutjesdijk et al. 1999;
Sivaraman et al. 2004), and B. carinata (Velasco et al. 2003). Additionally,
germplasm with increased erucic acid content, advantageous for non-food
applications, has been developed in B. carinata (Velasco et al. 1998).
5.5.2 Meal Quality

Seed glucosinolates are the major factor limiting the value of the meal for animal
feed. Germplasm with reduced levels of glucosinolates was developed in summer
(Downey et al. 1969) and winter types of B. rapa (Jönsson et al. 1975) as well as in
B. juncea (Love et al. 1990), which in combination with previously developed
zero-erucic acid types led to the development of double-zero cultivars of both
species. The first double zero cultivar of B. rapa, Candle, was registered in
Canada in 1997. The first double zero cultivars of B. juncea, Arid and Amulet,
were registered in Canada in 2002. Double zero cultivars of B. carinata have not
been developed yet, even though some progress is being made in the reduction of
seed glucosinolate content in this species (Márquez-Lema et al. 2006).
5.6.1 Seed Yield and Adaptation
Adaptation to growing conditions is an important objective in breeding pro-

grammes, especially in areas under risk of drought or frost. Despite the great
yielding potential of current cultivars of B. napus, especially the novel hybrid
cultivars, crop yields are surpassed by other Brassica crops under certain areas
due to a better adaptation to local environments. In many cases, comparative
trials have involved B. napus commercial cultivars versus landraces or germ-
plasm accessions of the other species not subject to modern breeding techni-
ques, which emphasizes the great potential for alternative Brassica spp. under
certain areas.
Spring-type cultivars of B. rapa are better adapted than B. napus cultivars to
short season growing areas (for example due to early spring or fall frosts), because
of their early maturity. Early maturity is also an advantage when the growing
season is limited by the occurrence of drought. B. carinata and B. juncea are more
resistant to drought than B. rapa and B. napus. A comparative evaluation of the
performance of B. carinata and B. napus in southern Spain concluded that the

better performance of B. carinata was produced by a faster canopy development
early in the season, combined with a longer flowering and grain filling period in
the presence of drought and high air temperatures (Fereres et al. 1983).
B. carinata has also been found to be better adapted than B. napus under warm
and droughty conditions during grain development in regions of the United
States (Knowles et al. 1981), Canada (Getinet et al. 1996; Warwick et al. 2006),
India (Malik 1990), and Italy (Mazzoncini et al. 1993). B. juncea is also better
adapted than B. napus to low rainfall areas, with the additional advantage over
B. carinata of earlier maturing (Getinet et al. 1996; Burton et al. 2007).
5.6.2 Vernalization Requirements and Flowering Time
Brassica rapa includes annual and biennial forms, the latter requiring vernalization,
i.e. the promotion of flowering in response to a prolonged period of growth at low
temperatures. High vernalization requirements are needed for fall planting in cold
climates, but they are undesirable for the development of early-maturing, spring
sown cultivars. Studies in this species have identified three loci controlling verna-
lization-responsive flowering time and three additional loci controlling flowering
time that are not responsive to vernalization (Osborn et al. 1997).
5.6.3 Male Sterility

Heterosis breeding has played a major role in recent years in increasing crop
yields in B. napus. Similarly, exploitation of heterosis is probably the best
approach to increase grain yield in the other Brassica oilseed crops. Accord-
ingly, extensive research has been conducted to develop male-sterility systems
suitable for commercial hybrid production. A number of sources of genetic or
nuclear male sterility (NMS) have been identified in B. rapa and B. juncea
(Pandey et al. 1999). Similarly, several cytoplasmic male sterility (CMS) and
fertility restoration systems have been developed in B. juncea (Pandey et al.
1999), some of them being further transferred to B. rapa and B. carinata.
However, in most cases the CMS systems were not suitable for commercial
production. Sodhi et al. (2006) have reported a new CMS system initially
identified in B. napus and transferred to B. juncea, with the particularity that
restoration was provided by any line different to the maintainer. The CMS
system, designated 126-1, is expected to be present in commercial hybrids in the
short term (Sodhi et al. 2007).
5.6.4 Self-Compatibility
The diploid species of Brassica are in general self-incompatible. The self-
incompatibility system is mainly controlled by a single locus S, which includes
three highly polymorphic genes that are transmitted as one segregational unit
(Sakamoto and Nishio 2001). There are several self-compatible lines in B. rapa.
In the cultivar Yellow Sarson, self-compatibility is controlled by two loci, S
(non-functional haplotype) and M, the latter being independent but epistatic to
S (Fujimoto et al. 2006).
5.6.5 Seed Colour, Oil, Protein and Fibre Content
Seed colour variations are common in B. rapa, B. juncea and B. carinata, with
genotypes ranging from dark brown to reddish-brown and from yellow-brown
to yellow. Yellow-coated seeds have higher oil and protein contents and lower
fibre content, which is attributable to a lower proportion of seed coat (Daun
and DeClercq 1988). Selection for yellow seed coat is therefore an important
breeding objective. In the three species, the trait has been found to be maternally
inherited. Most studies on B. rapa and B. juncea have concluded that yellow
seeds are the result of recessive alleles at two loci (Padmaja et al. 2005; Rahman
et al. 2007). In B. carinata, Getinet and Rakow (1997) proposed that yellow seed
coat was produced by a dominant repressor gene which inhibits the expression
of seed coat pigment synthesis genes.
5.6.6 Oil Quality
Oil quality is largely determined by the fatty acid composition and the presence of
minor compounds such as tocopherols and phytosterols affecting the nutritional
and/or technological properties of the oil. Different breeding strategies have
conducted to the development of modified fatty acid profiles, which have been
summarised above in the section of major breeding achievements. Erucic acid
content is controlled by multiple additive alleles at one locus in B. rapa (Dorrell
and Downey 1964) and at two loci in B. juncea (Kirk and Hurlstone 1983) and
B. carinata (Getinet et al. 1997). High oleic acid content (>80%) is under
monogenic control in B. rapa (Tanhuanpää et al. 1996b) and digenic control in
B. carinata (Nabloussi et al. 2006). Mid oleic acid levels (>70%) in B. juncea have
been obtained by antisense suppression of the fad2 gene (Sivaraman et al. 2004).
Research on minor oil components has been scarce in Brassica species other
than B. napus. Goffman et al. (1998) evaluated tocopherol content and profile in
several Brassica spp., concluding significantly higher concentrations of alpha-
tocopherol in B. carinata accessions. The tocopherol profile has been modified
in transgenic lines of B. juncea, which exhibited a concentration of 62% alpha-
tocopherol and 36% gamma-tocopherol, compared to 10% alpha-tocopherol
and 87% gamma-tocopherol in the untreated control (Yusuf and Sarin 2007).
5.6.7 Seed Meal Quality

Breeding for low glucosinolate content has been the principal objective in
relation to seed meal quality in Brassica species. Progress in this field has been
mentioned above in the section of major breeding achievements. Genetic studies

on low glucosinolate content in B. rapa concluded monogenic inheritance for
glucobrassicin and progoitrin contents, which were independently inherited
(Kondra and Stefansson 1970). In B. juncea, Sodhi et al. (2002) concluded the
involvement of seven genes in the genetic control of total glucosinolate content.
Other important breeding objectives such as the reduction of phytates and
phenolic compounds have been scarcely addressed in Brassica crops, except in
B. napus. Zhao (2007) has recently identified variation for low phytate content
in B. rapa, which opens up the possibility of breeding for this trait.
5.6.8 Disease Resistance

Some diseases cause problems in most growing areas, while others are restricted
to certain areas. The most important fungal diseases affecting Brassica crops
are Sclerotinia (Sclerotinia sclerotiorum), blackleg (Leptosphaeria maculans),
white rust (Albugo candida), Alternaria blight (Alternaria spp.), and downy
mildew (Peronospora parasitica). Brassica rapa is very susceptible to these
diseases. Additionally a root rot complex, known as brown girdling root rot
complex, caused primarily by Rhizoctonia solani, causes problems to this crop in
Canada (Soon et al. 2005). B. juncea is susceptible to most of the diseases,
although it shows a high degree of resistance to blackleg (Sacristan and Gerde-
mann 1996). White rust, downy mildew, and Alternaria are the more severe
diseases of this crop in India (Sangeetha and Siddaramaiah 2007). B. carinata
has a broad resistance to most of the diseases affecting the other Brassica crops
(Anand et al. 1985; Sacristan and Gerdemann 1996; Choudhary et al. 2000).
Breeding for resistance to major diseases is one important objective in
B. rapa and B. juncea. In B. rapa, sources of resistance to blackleg (Falak
et al. 1999; Leflon et al. 2007), Sclerotinia (Falak et al. 2006), white rust
(Tanhuanpää and Vilkki 1999a), Alternaria blight (Choudhary et al. 2000),
and brown girdling root rot (Woods et al. 2000) have been developed. In
B. juncea, white rust resistance has been introgressed from B. napus (Somers
et al. 2002). Some Australian germplasm of this species has been found to show
some degree of tolerance to Sclerotinia (Singh et al. 2007). Development of
Alternaria blight resistance in B. juncea has not been achieved through conven-
tional breeding, but there is promising ongoing research to develop resistant
sources through transgenic research (Mondal et al. 2007). Resistance to downy
mildew is available in Indian germplasm (Yadava and Singh 1999).
5.6.9 Insect Resistance

The mustard aphid (Lipaphis erysimi) is one of the most important pests of
oilseed brassicas, causing severe yield losses particularly in India (Patel et al.
2004). B. carinata has less susceptibility to aphid infestations than B. rapa and
B. juncea (Malik 1990; Rana 2005). Resistance to the mustard aphid in B. juncea
was achieved by transformation with a cDNA encoding different types of
agglutinins (Kanrar et al. 2002; Hossain et al. 2006).
Breeding strategies are strongly determined by the mode of reproduction of the

different species. In the partially allogamous species B. juncea and B. carinata,
pedigree selection and backcrossing have been widely used to develop line
cultivars, although breeding of improved populations as ‘synthetic cultivars’
has also been considered (Becker et al. 1999). In the self-incompatible species
B. rapa, breeding procedures suitable for partially allogamous species are not
appropriate, with the exception of backcrossing. For this species, various types
of recurrent selection have been efficient to develop high yielding cultivars
(Downey and Rakow 1987). The development of hybrid cultivars as a means
of full exploitation of heterosis can be used in the three species.
5.7.1 Developing New Sources of Variation
Breeding programs deal with the manipulation of naturally existing genetic

variation available through germplasm collections or newly generated varia-
tion. The amount of natural genetic variation available to the Brassica oilseed
breeder, considering all the species of the genus, is impressive. The first step in
searching for novel traits is the evaluation of intraspecific variation existing in
local varieties and landraces. Although the within-species variability is often
insufficient for many traits, there are examples of identification of variation
through the evaluation of genetic resources within the same species, such the
isolation of erucic acid-free lines of B. rapa (Downey 1964), B. juncea (Kirk and
Oram 1981) and B. carinata (Alonso et al. 1991), or lines with high oil content
(De Haro et al. 1998) or low linolenic acid content in B. carinata (Velasco et al.
1997a).
A further approach for incorporating desired traits that are not present in the
crop species is the hybridization between related species. Interspecific hybridi-
zation with other species within the triangle of U has been used to eliminate
erucic acid in B. carinata (Fernández-Martı́nez et al. 2001; Getinet et al. 1994)
or to develop low glucosinolate types in B. juncea (Love et al. 1990). Interge-
neric hybridisation has been widely used to develop CMS systems (Pandey et al.
1999; Sodhi et al. 2006). Genetic engineering is being increasingly used to
develop novel variation for a number of traits, which is reviewed below.
Mutagenesis has been successfully used to generate genetic variation for
useful traits, particularly seed oil quality traits, in Brassica spp. In B. carinata,
mutants with reduced (Velasco et al. 1995) and increased (Velasco et al. 1998)
levels of erucic acid, as well as mutants with altered levels of unsaturated fatty
acids (Velasco et al. 1997b) were obtained using chemical mutagenesis.
In Indian mustard, Oram and Kirk (1993) also reported induced mutants
with reduced linolenic acid content, whereas Bhat et al. (2001) developed
CMS-induced mutants.
5.7.2 Breeding of Line and Population Cultivars

For the amphidiploid partially allogamous species B. juncea and B. carinata,
although they always undergo some outcrossing, the most commonly used
breeding method has been pedigree breeding to develop line cultivars. The
first step involves the selection of parents to be crossed after a careful evaluation
of the traits to be combined, followed by artificial hybridization. After crossing,
selfing is continued during seven to eight successive generations. Selection is
made each generation to combine the desired traits such as low erucic acid and
low glucosinolate content, high yield, and disease resistance, in stable uniform
lines. The best homogeneous F6 or F7 plots are used to obtain breeder´ s seed of
new candidate cultivars (Downey and Rakow 1987; Becker et al. 1999).
Alternatives to the pedigree method are the single seed descent (SSD) and the
development of doubled haploids (DH) lines. The latter abbreviates the time
required to achieve homozygosity. Haploids can be induced in large amounts by
culturing microspores on appropriate nutrient media to produce plantlets, and
then the seedlings are induced to chromosome doubling with colchicine treatment.
A simplified alternative method is based on microspore colchicine treatment in
vitro. This technique has been widely adopted by B. juncea breeders (Sodhi et al.
2002), but it has been more sporadically used in B. carinata (Chuong and
Beversdorf 1985) and B. rapa (Seguin-Swartz et al. 1983).
Backcrossing is a breeding method that has been also successfully used in
cultivated Brassica species to transfer simply inherited traits such as low erucic
acid or low glucosinolate content to adapted high-yielding lines. The proce-
dures used to transfer the zero erucic trait to B. juncea and B. carinata using this
method are the same described for B. napus (Downey and Rakow 1987), since
the genetic control of zero erucic content in the three species is similar: embryo-
nic control and two loci with multiple alleles having an additive effect (Kirk and
Hurlstone 1983; Getinet et al. 1997). In the diploid B. rapa, erucic acid is
controlled at a single locus (Dorrell and Downey 1964). The heterozygous
seeds E1e1E2e2 (E1e1 in the case of B. rapa) are selected in the F1 or BC1F1
generations, which enables to make a backcross per generation, thus speeding
up the transfer of the trait. In the case of recessive traits that do not show up in
the F1 generation, for example the high oleic acid trait of B. carinata controlled
by two recessive genes (Nabloussi et al. 2006), the use of marker-assisted
selection strategies allowing an efficient identification of heterzygotes facilitates
the introgression. An interesting discussion on the value of the backcross

method in combination with marker-assisted selection for traits with complex
inheritance such as total glucosinolate content can be found in Ramchiary et al.
(2007b). Depending on the objectives, a combination of backcrossing and
pedigree selection is frequently used (Downey and Rakow 1987). This is parti-
cularly useful when the breeder wishes to combine simply inherited traits such
as low erucic acid with more complex traits such as high oil content and high
seed yield.
In the cross-pollinated self-incompatible forms of B. rapa, the standard
breeding method is recurrent selection. This method, described in detail by
Downey and Rakow (1987) and Dhillon et al. (1995), starts with the planting
of spaced plants from source populations (landraces, progenies from crosses,
etc.). These plants are harvested individually and a portion of the seeds from
each plant is used for progeny row evaluation. The seed of selected rows are
further evaluated for quality traits and the reserve seed from selected single
plants is composited and planted under isolation to allow random mating and
form a new population that will enter a new cycle. The selection is continued for
at least three cycles. Another procedure used in B. rapa has been the production
of synthetics by mixing two or three parental components (Becker et al. 1999).
This method was even suggested for the partially allogamous species B. juncea
and B. carinata to make a partial use of the heterosis. However, at present most
of the efforts on utilization of heterosis are directed to produce F1 hybrid
cultivars.
5.7.3 Breeding of Hybrid Cultivars
As in B. napus, the exploitation of the available heterosis in the other oilseed

brassicas can be used for enhancing productivity. Many studies have shown
significant levels of heterosis in these species, for example in B. juncea (Pradhan
et al. 1993), B. rapa (Falk et al. 1994), and B. carinata (Teklewold and Becker
2005). The interest in developing hybrid cultivars has gained importance in
recent years, as effective systems of male sterility to control pollination have
been developed. The utilization of genetic or nuclear male sterility (NMS)
available in B. juncea and B. rapa presents economical limitations due to the
high labour costs involved to rouge out the male fertile plants in the seed
production fields before flowering. These difficulties have been overcome
through the development of cytoplasmic male sterility (CMS) and fertility
restoration systems. These systems permit the full exploitation of heterosis in
F1 hybrids by the relatively inexpensive means of producing male-sterile female
(A), maintainer (B) and restorer (R) lines. In B. juncea, a number of CMS
sources have been obtained (reviewed in Pandey et al. 1999 and Sodhi et al.
2006), but the lack of appropriate restorer lines has hampered their exploitation
for producing commercial hybrid seed (Sodhi et al. 2006). However, a new CMS
named 126-1 was recently reported to be suitable for hybrid production in

B. juncea (Sodhi et al. 2006), and a hybrid cultivar based on this CMS source
has been produced and tested with success over a large area in the mustard
growing region of India (Sodhi et al. 2007). In B. rapa, although the potential of
CMS-based hybrids has been experimentally demonstrated (McVetty 1995),
commercial developments have been less successful. No reports on hybrid
cultivars have been published for B. carinata.
After effective systems for controlled pollination are available, the most
important task is the optimization of all steps of the method to identify the
best hybrid combinations. The selection of the best cross combinations can be
made in three steps (Becker et al. 1999): selection among the components for
their per se performance, selection among the test crosses for general combining
ability, and selection among the hybrids for both general and specific combin-
ing ability.
5.7.4 Breeding Techniques
The following techniques, used for greenhouse and field nurseries as well as for
seed quality evaluation, are usually considered in oilseed mustard and turnip
rape breeding programs.
5.7.4.1 Selfing and Artificial Hybridization

In the partially allogamous species B. juncea and B. carinata, self-pollinated
seed is obtained by enclosing one or more racemes free of open flowers with
paper or microperforated plastic bags. In all the species, intraspecific crosses are
performed by emasculating flower buds that are about to open or will open
within the following day, by removing with tweezers the six undehisced anthers.
Pollination is accomplished by picking selected anthers with forceps and apply-
ing them to the stigma of emasculated flowers. In the field nurseries, pollen is
often applied by dusting emasculated stigmas with racemes of male flowers
gathered in the early morning and stored with their stems in water. Fertilization
occurs normally within 24 h of pollination, but protective bags should remain in
place for at least 3 days. For interspecific hybridization, the success rate
depends on the direction of the cross. In crosses involving diploid and amphi-
diploid species, more seed set is observed when the amphidiploid species is used
as the female parent (Downey and Rakow 1987).
5.7.4.2 Techniques Used for Agronomic Evaluation

Newly developed cultivars have to be carefully evaluated before they can be
released. For early-generation testing, each genotype is usually sown in single-
row plots with check cultivars intercalated every five to ten rows. For breeding
material undergoing precise evaluation, four to six-row plots are used. Phenolo-
gical and morphological data are recorded in the field. At harvest, seed yields of
each plot are recorded and a subsample is taken for analysis of seed quality traits.
5.7.4.3 Laboratory Techniques for Seed Quality Evaluation

Success in the improvement of seed quality traits in oilseed brassicas has been
facilitated by an important previous research on novel analytical methods and
techniques suitable for screening purposes, but still reliable enough. The half-seed
technique was developed for the analysis of the fatty acid profile in part of the
cotyledonary tissue without affecting the germination ability of the rest of the
seed (Downey and Harvey 1963). The use of near-infrared reflectance spectro-
scopy (NIRS) has contributed to the success of breeding programs focusing on
seed quality. The technique is non-destructive and used for the simultaneous
analysis of a wide range of seed quality traits in Brassica seeds, including oil,
protein, glucosinolates, fibre, fatty acids, bulk density, thousand seed weight, and
others (Velasco and Fernández-Martı́nez 2002). An example of the value of
NIRS in breeding programs was its application in a mutagenesis program of
B. carinata that contributed to broaden the genetic variation of this species for
different traits, particularly for seed oil fatty acid profile (Velasco et al. 1995,
1997b, 1998). The technique has also been adapted to single-seed analysis for
fatty acid profile in B. carinata. Velasco et al. (2003) reported its application in a
breeding program aimed at developing high oleic acid content in this species.
The development of molecular tools to assist in selection and to develop novel

genetic variation has represented a major landmark in Brassica oilseed breeding.
Brassica crops benefit from the close phylogenetic relationship with Arabidopsis,
the model plant for forefront research on plant molecular genetics. Because of its
diploid genome shared by B. napus, research on B. rapa was particularly intensive
in the first stages of molecular breeding research, both on vegetable and oilseed
types. Important efforts on B. juncea molecular research have been also made,
especially in recent years. The situation of B. carinata is completely different,
since there are few research groups with molecular and biotechnological facilities
conducting research on this species. Because of the high degree of sequence
conservation within the genus, research on other Brassica species, especially
B. nigra and B. oleracea, is also of great utility for the oilseed Brassica species.
5.8.1 Genetic Markers and Genetic Linkage Maps

Like in other species, the first genetic linkage maps on Brassica species were
produced using restriction fragment length polymorphism (RFLP) markers.
The first genetic map of B. rapa was developed for vegetable types (Song et al.
1991). The first RFLP map of oilseed B. rapa was based on 360 marker loci
distributed in 10 linkage groups, which is the haploid chromosome number of
the species (Chyi et al. 1992). In B. juncea, the first RFLP map with good
genome coverage was produced by Cheung et al. (1997), consisting of 343 loci
arranged in 18 major linkage groups, which is the haploid chromosome number
of the species.
A second generation of genetic linkage maps was produced by using
PCR-based markers, mainly random amplified polymorphic DNA
(RAPD) and amplified fragment length polymorphism (AFLP) markers.
Tanhuanpää et al. (1996a) developed a genetic map of B. rapa using 22
RFLP and 144 RAPD markers, distributed over the 10 linkage groups. In
B. juncea, Sharma et al. (2002) developed a genetic map with 130 RAPD
markers arranged in 21 linkage groups, whereas Lionneton et al. (2002)
constructed a map based on 264 AFLP and 9 RAPD markers distributed in
18 linkage groups. RAPD, AFLP, and inter simple sequence repeat (ISSR)
bands can be converted into more reproducible sequence-characterized
amplified region (SCAR) markers, which have been developed for B. rapa
(Tanhuanpää and Vilkki 1999b) and B. juncea (Ripley and Roslinsky 2005;
Ashutosh et al. 2007).
The use of microsatellites or simple sequence repeats (SSRs) represented a
further advance in molecular breeding due to their extraordinary level of infor-
mative polymorphism, repeatability, and amenability to automation. Microsa-
tellites have been mainly developed from the diploid species of Brassica, including
B. rapa, and from B. napus (Suwabe et al. 2002; Lowe et al. 2004). Because of the
high transferability of SSR markers across related Brassica species, SSR markers
developed from B. rapa, B. oleracea, B. nigra and B. napus have been used for
molecular research and breeding in B. juncea (Ramchiary et al. 2007a) and
B. carinata (A. Márquez-Lema, personal communication).
Expressed sequence tags (ESTs) constitute a novel source of DNA-based
markers that are physically associated with coding regions of the genome.
Several types of markers based on ESTs have been developed for Brassica
species, for example EST polymorphisms (ESTP) (Choi et al. 2007) and
EST-based RFLPs (Kim et al. 2006) in B. rapa, and EST-SSRs in B. juncea
(Hopkins et al. 2007).
High-density linkage maps have been developed for both B. rapa and
B. juncea. In B. rapa, a map comprising a total of 556 markers, including 278
AFLP, 235 SSR, 25 RAPD, and 18 sequence-based markers, covering ten
linkage groups, was developed. The total length of the linkage map was
1,182 cM, with an average marker interval of 2.8 cM (Choi et al. 2007). Another
linkage map of this species was constructed using 545 sequence-tagged loci,
mainly EST-based RFLPs. The map covered 1,287 cM, with an average map-
ping interval of 2.4 cM (Kim et al. 2006). In B. juncea, a high density linkage
map included 996 AFLP and 33 RFLP markers, aligned in 18 linkage groups.
The total map length was 1,629 cM, with an average interval of 3.5 cM between
adjacent loci (Pradhan et al. 2003).
5.8.2 Molecular Breeding
5.8.2.1 Germplasm Characterization

In the genus Brassica, molecular markers have been used for determination of
phylogenetic relationships (Song et al. 1988) as well as for identification of
genetic variation present in germplasm collections. Studies on genetic diversity
among germplasm accessions based on molecular markers have been conducted
in B. rapa (Zhao et al. 2005), B. juncea (Burton et al. 2004), and B. carinata
(Teklewold and Becker 2006; Warwick et al. 2006).
5.8.2.2 Molecular Mapping

Seed Colour
Molecular markers linked to loci controlling seed coat colour have been devel-
oped in B. rapa and B. juncea. In B. rapa, Teutonico and Osborn (1994) mapped
the recessive gene Yls controlling yellow seed colour using RFLP markers. In
B. juncea, Sabharwal et al. (2004) and Padmaja et al. (2005) identified AFLP
and SSR markers, respectively linked to two independent loci controlling seed
coat colour, whereas Mahmood et al. (2005) detected three QTL that individu-
ally explained 43%, 21%, and 16%, respectively, of the phenotypic variation
for this trait.
Oil Content and Quality

Several studies have identified QTL affecting seed oil content in B. juncea.
Lionneton et al. (2002) identified two QTL for this trait in an evaluation
under a single environment. Ramchiary et al. (2007a) evaluated a doubled
haploid mapping population under three environments, identifying two QTL
affecting oil content in all the three environments, one QTL affecting oil content
in one environment, whereas other four QTL were identified in single
environments.
Extensive research has been conducted to develop molecular markers linked
to genes controlling fatty acid biosynthesis and to cloning and sequencing such
genes. In B. rapa, an SCAR marker linked to a locus for high oleic acid content
was developed (Tanhuanpää et al. 1996b). The locus was identified as the
omega-6 fatty acid desaturase (fad2) gene, which enabled the development of
fad2 allele-specific markers (Tanhuanpää et al. 1998). One major QTL for
increased oleic acid content was identified in high erucic acid B. juncea (Sharma
et al. 2002). In B. rapa, Tanhuanpää and Schulman (2002) identified three QTL
affecting linolenic acid content in a population segregating for low linolenic
acid levels. One of the QTL was identified as the omega-3 fatty acid desaturase
(fad3) gene through a candidate gene approach. Gupta et al. (2004) identified
two QTL underlying the variation for erucic acid content in a population
developed from a zero and a high erucic acid line. The QTL were associated
with two fatty acid elongase 1 (FAE1) genes. The authors identified seven single
nucleotide polymorphisms (SNPs) between the sequences of the genes in high
and low erucic acid lines. In B. rapa, Teutonico and Osborn (1994) mapped the
gene controlling erucic acid content on an RFLP linkage map.
Glucosinolate Content
Molecular markers associated with seed glucosinolate content have been identified
in crosses involving low and high glucosinolate cultivars of B. juncea. Mahmood
et al. (2003) identified two QTL associated with gluconapin (3-butenyl) content,
three QTL affecting sinigrin (2-propenyl), and five QTL associated with total
glucosinolate content. Interestingly, the major QTL associated with individual
glucosinolates were not significant for total glucosinolate content. Ripley and
Roslinsky (2005) developed an SCAR marker linked to high sinigrin content
after conducting bulked segregant analysis with ISSR markers. Lionneton et al.
(2004) used a population developed from crosses of two lines with high glucosi-
nolate content but different glucosinolate profiles, identifying two consistent QTL
for both sinigrin and gluconapin content.
Morphological and Agronomic Traits

Several studies have identified QTL consistently associated with morphological
and agronomic traits. Teutonico and Osborn (1994) mapped one locus control-
ling presence or absence of leaf hairs in B. rapa. Lionneton et al. (2004) found
two distinct QTL for plant height in B. juncea. In the same crop, Mahmood et
al. (2007) identified three QTL for plant height that were significant, at least, in
two or three environments. Ramchiary et al. (2007a) conducted QTL analysis in
a B. juncea population segregating for 12 agronomic traits such as plant height,
number of primary and secondary branches, siliquae per plant, seeds per
siliqua, etc. The authors identified 65 QTL for these traits using data from
three environments, nine of them detected in all the three environments.
Male Sterility
A stable CMS line of B. juncea was developed through somatic hybridization
with Moricandia arvensis, which was also the genetic source for fertility restora-
tion (Prakash et al. 1998). Ashutosh et al. (2007) identified two AFLP and one
SCAR markers linked to the fertility restoration locus Rf.
Vernalization Requirements and Flowering Time

Studies in B. rapa have identified three QTL controlling vernalization-responsive
flowering time and three additional QTL controlling flowering time that are not
responsive to vernalization (Osborn et al. 1997). In B. juncea, which has no
vernalization requirements, Ramchiary et al. (2007a) identified four QTL affect-

ing flowering time, two of them expressed over three contrasting environments
and the other two QTL expressed in two environments. Mahmood et al. (2007)
identified five QTL affecting days to first flowering considering the average of
four environments. One of the QTL was also significant in all the four individual
environments, and it was tightly linked to QTL affecting days to end of flowering,
days to maturity, and also plant height.
Disease Resistance
White rust caused by Albugo candida is one of the most important diseases of
B. rapa and B. juncea. A. candida is classified into ten races based on host
specificity. B. juncea is infected by race 2, whereas B. rapa is infected by race 7,
although there is variation for virulence between biotypes of the same race. Most
studies have shown that resistance to both races is governed by single dominant
genes (Tanhuanpää 2004). Several independent loci controlling resistance to
white rust have been mapped in B. rapa (Kole et al. 2002; Tanhuanpää 2004)
and B. juncea (Cheung et al. 1998; Somers et al. 2002; Varshney et al. 2004).
Blackleg is the most important disease affecting B. rapa worldwide. At least
five resistance genes have been identified in B. rapa germplasm. Two of them,
Rlm1 and Rlm7 have been mapped to the R7 linkage group of this species
(Leflon et al. 2007). Two resistance genes identified in B. juncea, LMJR1
(dominant) and LMJR2 (recessive), have been mapped in the B genome of
this species (Christianson et al. 2006).
5.8.3 Marker Assisted Selection
The availability of molecular markers tightly linked or based on the sequences

of genes of interest is a powerful tool for selection based on the genotype rather
than the phenotype of the plants. The accuracy of marker assisted selection
largely depends on the number of genes involved and the proximity of the
markers to the gene. For erucic acid content, which is a trait controlled by
additive alleles at two loci, allele-specific SNP markers have been developed in
B. juncea, which ensures an accurate tagging of the trait in marker assisted
backcross programs (Gupta et al. 2004). Similarly, Tanhuanpää et al. (1998)
developed an allele-specific marker in B. rapa based on the sequence of the fad2
locus, which encodes the enzyme responsible for the desaturation of oleic acid
to linoleic acid.
The opposite situation is the selection for complex genetic traits, such as
glucosinolate content, based on molecular markers identified through QTL
analysis and not based on the sequences of the genes. In this case, the existence
of epistatic and context dependent interactions may considerably reduce the
selection efficiency (Ramchiary et al. 2007b).
5.8.4 Transgenic Breeding
Transgenic technologies are increasingly being used in Brassica oilseed breed-

ing. Much of the research is being conducted in B. juncea. One of the main
objectives has been to modify the fatty acid profile of the seed oil. In this
research area, transgenic B. juncea plants with increased levels of oleic acid
and reduced levels of linolenic acid (Stoutjesdijk et al. 1999; Sivaraman et al.
2004; Jha et al. 2007), or with reduced levels of total saturated fatty acids in the
seeds (Yao et al. 2003) have been developed. Genetic manipulation of this crop
has also resulted in the production of transgenic plants with ability to accumu-
late gamma-linolenic acid in the seeds (Hong et al. 2002; Das et al. 2006). The
tocopherol fraction of the seeds has been also modified through a transgenic
approach. Yusuf and Sarin (2007) produced transgenic lines with enhanced
accumulation of alpha-tocopherol by overexpressing a gamma-tocopherol
methyl transferase cDNA.
Transgenic research has been applied to the fields of disease and insect
resistance. Mondal et al. (2007) enhanced resistance to Alternaria blight in
B. juncea by transformation with a glucanase protein from tomato. Resistance
to the mustard aphid has been achieved through transgenic approaches (Kanrar
et al. 2002; Hossain et al. 2006). Herbicide resistance is another trait for which
transgenic B. juncea lines have been developed (Green and Salisbury 1999;
Mehra et al. 2000).
In recent years, there is a considerable interest in using B. juncea for phytor-
emediation of contaminated soils. There are a number of transgenic lines of
B. juncea with improved ability to accumulate toxic metals and metalloids
(Gasic and Korban 2007) or organic pollutants (Flocco et al. 2004).
5.9 Seed Production
The rapeseed and mustard breeder identifies pure lines or populations that
perform better than the currently used cultivars. The seed of this material,
referred to as breeder seed or prebasic seed, is maintained and increased by
the breeder and it is genetically the purest source of a cultivar. When applied to
hybrid cultivars, it refers to the seed of male-sterile (A), maintainer (B) and
restorer (R) lines. The initial increase of breeder seed, referred to as foundation
seed, is also grown, directly or indirectly, under the supervision of the plant
breeder. Registered seed is the progeny of breeder and foundation seed handled
under procedures acceptable to the certifying agency to maintain satisfactory
genetic purity and identity. Certified seed is the progeny of breeder and founda-
tion seed.
The increase and production of breeder, foundation, and certified seed is
critical and requires highly qualified seed producers. The standards of purity,
identity and certification procedures vary from country to country (Downey
and Rakow 1987). For example, the levels of uniformity required in Europe are
greater than for other producing regions of the world. The requirements for
cultivar uniformity vary also with the species, being less stringent for cross-
pollinated B. rapa populations.
The increase of breeder/foundation seed of pure line cultivars, as well as
A, B and R lines in the case of hybrid seed production, must be accom-
plished under spatial isolation or using screened cages. For spatial isola-
tion, McVetty (1995) recommended in Canada an isolation distance of 800
m and the R line planted 2 m around the hybrid seed production field. In
China, Dianrong (1999) recommended an isolation distance of over 2,000
m. The planting ratio (female:male) is also a crucial aspect for an econom-
ical hybrid seed production. Depending on the CMS source, Yadav and
Chakrabarty (2007) suggested female:male ratios varying from 8:2 to 16:2
in B. juncea. Two hives of honeybees per hectare are placed within the
maintainer and hybrid seed production field during the pollinating period
(Dianrong 1999).
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Chapter 6
Sunflower
José M. Fernández-Martı́nez, Begoña Pérez-Vich, and Leonardo Velasco
6.1 Introduction
Sunflower (Helianthus annuus L.) oil is the fourth most important vegetable oil
in world trade at present with an annual production of around 9 million tonnes
and a cultivated acreage of over 22 million hectares, mainly concentrated in the
Russian Federation, Ukraine, India, and Argentina, which totalize more than
50% of sunflower world acreage. Although of North American origin, where it
was domesticated by the Native American Indians for its edible seeds (Heiser
et al. 1969), the transformation of sunflower into a major oilseed crop only took
place in the second half of the 20th century due to two major breeding achieve-
ments: the drastic increase of oil percentage in sunflower achenes achieved in
the Former Soviet Union from 1920 to 1960 (Gundaev 1971), and the develop-
ment of a cytoplasmic male sterility system (Leclercq 1969) combined with
fertility restoration by nuclear genes (Kinman 1970) that enabled the commer-
cial production of hybrid seed. The subsequent development of short-stemmed,
high yielding hybrid cultivars with high oil content well adapted to mechanised
cropping represented the transformation of sunflower into a cash crop and
sunflower oil into a major commodity in world trade.
Conventional sunflower produces a healthful oil with great consumer
acceptance because of its high content of monounsaturated and polyunsatu-
rated fatty acids as well as high vitamin E content. In recent years, new
sunflower oil types for specific applications, mainly in the food industry,
have been developed through conventional breeding approaches. Such speci-
alty oils are called to play an important role in a further development of the
sunflower crop.
Unlike other oilseed crops such as soybean and canola, commercial sun-
flower has not been subject to transgenic breeding so far. However, sunflower
breeders have been very successful in attaining a wide diversity of breeding
objectives, from developing novel seed oil quality types to incorporating genetic
J.M. Fernández-Martı́nez (*)

Institute for Sustainable Agriculture (CSIC), Córdoba, Spain
e-mail: cs9femaj@uco.es

156 J.M. Fernández-Martı́nez et al.
resistance to most of the pests and diseases that threaten the crop. The history,
current status, and future prospects of breeding advances in sunflower are
reviewed in this chapter.
The cultivated sunflower (Helianthus annuus L.) is a member of the family

Compositae (Asteraceae). The basic chromosome number is n = 17. The genus
includes diploid, tetraploid and hexaploid species. The closest relatives appear to
be Tithonia, Viguiera and Phoebanthus (Heiser et al. 1969). The common sun-
flower (H. annuus) is the most important species grown commercially, although
other species are also cultivated, e.g. H. tuberosus, which is grown for production
of edible tubers, and several other species grown as ornamentals. The name
Helianthus is derived from the Greek words ‘‘helios,’’ meaning sun, and ‘‘anthus,’’
meaning flower. The Spanish name for sunflower, ‘‘girasol,’’ and the French
name ‘‘tournesol’’ literally mean ‘‘turn with the sun,’’ a trait exhibited by sun-
flower until anthesis, after which the capitula (heads) face east.
Heiser et al. (1969) proposed a species classification of the genus Helianthus
including 14 annual and 36 perennial species from North America (in three
sections and seven series) and 17 species from South America. More recent
classifications (Schilling and Heiser 1981; Jan and Seiler 2007) have introduced
some modifications. The new classification brings the number of species to 51,
with 14 annual and 37 perennial species (Tables 6.1 and 6.2).
Prior to the arrival of the European explorers to the New World, the
progenitor of cultivated sunflower, the wild H. annuus was restricted to
the southern U.S. (Heiser 1978). Wild H. annuus was used for food by the
Native American Indians and, due to its association with humans, it became
a camp-following weed that was introduced into the central part of the U.S.,
where it was domesticated and carried to the east and southwest (Heiser et al.
1969). The earliest evidence of domesticated sunflower has been dated at
4,625 B.P. (Crites 1993).
The origin of cultivated sunflower has been also investigated using molecular
techniques. The use of randomly amplified polymorphic DNA (RAPD) mar-
kers supported the hypothesis that wild H. annuus was the progenitor of
cultivated sunflower (Arias and Rieseberg 1995). Further studies using allo-
zyme variation (Cronn et al. 1997) concluded that wild H. annuus from the
Great Plains include the most likely progenitor of domesticated sunflower.
More recent results based on the genetic relation between wild and extant
domesticates (Harter et al. 2004) support the hypothesis that extant domesti-
cated sunflowers arose from wild populations in the central part of the U.S.
Other investigations using QTL analysis have studied the identity of traits that
were the primary targets of strong selection during domestication (Burke et al.
2002). They concluded that strong directional selection for increased achene
size appears to have played a central role in sunflower domestication.
6 Sunflower 157
Table 6.1 Infrageneric classification of annual Helianthus species (n ¼ 17)

Sectiona Species
Helianthus H. annuus L.
H. anomalus S.F. Blake
H. argophyllus Torr.& A. Gray
H. bolanderi A. Gray
H. debilis Nutt.
subsp. debilis
subsp. cucumerifolius (Torr. & A. Gray) Heiser
subsp. silvestris Heiser
subsp. tardiflorus Heiser
subsp. vestitus (E. Watson) Heiser
H. deserticola Heiser
H. exilis A. Gray
H. neglectus Heiser
H. niveus (Benth.) Brandegee
subsp. canescens (A. Gray) Heiser
subsp. niveus
subsp. tephrodes (A. Gray) Heiser
H. paradoxus Heiser
H. petiolaris Nutt.
subsp. fallax Heiser
subsp. petiolaris
H. praecox Engelm. & A. Gray
subsp. hirtus (Heiser) Heiser
subsp. praecox
subsp. runyonii (Heiser) Heiser
Agrestes H. agrestis Pollard
Porteri H. porteri (A. Gray) Pruski
a
Schilling and Heiser (1981); Jan and Seiler (2007)
The domesticated sunflower was introduced from North America into

Europe by the early Spanish explorers in 1510 (Putt 1997), where they initially
gained popularity as a garden ornamental. The agronomic development of
sunflower as an oilseed crop and for use as edible achenes (confectionery
types) took place in Russia, where a number of landraces had been developed
by the late 1800s. Initial selection emphasis was given to early maturity, disease
and pest resistance, and high seed oil content. Sunflower was reintroduced from
Russia to North America in the latter part of the 19th century (Putt 1997).
6.3 Varietal Groups
There are three major groups of varieties of cultivated sunflower (H. annuus):
those used for the extracted seed oil (oilseed types), those for the direct con-
sumption of the seeds (confectionery types), and those used as ornamentals.
Table 6.2 Infrageneric classification of perennial Helianthus species

Chromosome
Sectiona Series Species number (n)
Ciliares Ciliares H. arizonensis R.C. Jacks. 17
H. ciliaris DC. 34,51
H. laciniatus A. Gray 17
Ciliares Pumili H. cusickii A. Gray 17
H. gracilentus A. Gray 17
H. pumilus Nutt. 17
Atrorubens Coronasolis H. californicus DC. 51
H. decapetalus L. 17,34
H. divaricatus L. 17
H. eggertii Small 51
H. giganteus L. 17
H. grosseserratus M. Martens 17
H. hirsutus Raf. 34
H. maximiliani Schrad. 17
H. mollis Lam. 17
H. nuttallii Torr. & A. Gray
subsp. nuttallii 17
subsp. parishii (A. Gray) Heiser 17
subsp. rydbergii (Britton) R. Long 17
H. resinosus Small 51
H. salicifolius A. Dietr. 17
H. schweinitzii Torr. & A. Gray 51
H. strumosus L. 34,51
H. tuberosus L. 51
Atrorubens Microcephali H. glaucophyllus D.M. Sm. 17
H. laevigatus Torr. & A. Gray 34
H. microcephalus Torr. & A. Gray 17
H. smithii Heiser 17,34
Atrorubens Atrorubentes H. atrorubens L. 17
H. occidentalis Riddell
subsp. occidentalis 17
subsp. plantagineus (Torr. & A. Gray) 17
Heiser
H. pauciflorus Nutt.
subsp. pauciflorus 51
subsp. subrhomboideus (Rydb.) O. Spring & 51
E.E. Schill.
H. silphioides Nutt. 17
Atrorubens Angustifolii H. angustifolius L. 17
H. carnosus Small 17
H. floridanus A. Gray ex Chapm. 17
H. heterophyllus Nutt. 17
H. longifolius Pursh 17
H. radula (Pursh) Torr. & A. Gray 17
H. simulons E. Watson 17
H. verticillatus Small 17
a
Schilling and Heiser (1981); Jan and Seiler (2007)
6 Sunflower 159
Hybrid varieties are nowadays predominant for all three groups. By far, the
major portion of sunflower production is devoted to oil extraction (Miller and
Fick 1997). Sunflower oil has been traditionally viewed as a healthful vegetable
oil and it is considered a premium oil for salad, cooking, and margarine
production. The seeds of confectionery sunflower varieties are used as snack
food as well as for feeding birds and small animals. The main characteristics
that differentiate oilseed and confectionery sunflowers are oil content and seed
size. The oilseed varieties have small black seeds with low hull content and very
high oil content (about 50%). Conversely, confectionery sunflower varieties
have larger seeds, which are usually black with white stripes, with lower oil
content (about 30%) and a higher hull percentage.
Oilseed sunflower varieties are divided into three groups according to their
oleic acid content: linoleic, mid-oleic, and high oleic. Linoleic (traditional)
varieties have linoleic acid content between 45 and 75%, depending on the
environment. It is considered a healthy vegetable oil suitable for salad and
margarine production. The seed oil of mid- and high-oleic varieties has an
oleic acid content of 55–75% and 85–90%, respectively. These oils are char-
acterized by a better thermooxidative stability, which makes them more appro-
priate for frying purposes.
The main criterion of quality of confectionery sunflower is the seed size
(Lofgren 1997). The largest size (>7 mm) type goes into the in-shell market to
be used as snack. Medium-size seeds are hulled for the kernel market and those
with the smallest size go into the bird and pet feeding market.
The last group of sunflower varieties includes those grown for ornamental
purposes. There is great diversity for floral colour (yellow, cream, orange, rose,
red, burgundy and bicolour) and morphology as well as for plant height (Fick
1976). Head diameter can vary from 10 to more than 30 cm and plant height
from 50 to 500 cm (Miller and Fick 1997). Ornamental sunflower cultivars are
used in gardens, home landscapes or as cut flowers. Cultivars used in home
gardens are usually classified in groups based on plant height, which include
giant or very tall (2.5–5 m), semi-dwarf (1–2.5 m), and dwarf types (<1 m). The
cultivars used as cut flowers are pollen-free types which incorporate the cyto-
plasmic male-sterility trait. Ornamental cultivars usually incorporate genes
from wild Helianthus spp.
Genetic resources are the base of crop improvement. They consist of the total
pool of variability that exists in the cultivated species and also in related species
that are sexually compatible with the cultivated one. Cultivated sunflower can
be crossed with most of the 51 Helianthus spp. Sunflower germplasm resources
can be categorized as ex situ resources (accessions preserved in seed banks) and
in situ resources (wild populations and land races).
6.4.1 Germplasm Collection and Maintenance
6.4.1.1 Ex Situ World Collections

Aggressive collection of wild and cultivated sunflower germplasm for preserva-
tion in seed banks is crucial to make it easily available to sunflower breeders.
Given the tenuous situation of some wild species in their natural habitats and
the replacement of local landraces by outstanding high-yielding improved
cultivars, seed banks may provide the only way to preserve these germplasm
resources for posterity.
Systematic collection, introduction and conservation of sunflower germ-
plasm were carried out by the N.I. Vavilov All-Union Scientific Research
Institute (VIR) at St. Petersburg, Russia and by the U.S. National Plant
Germplasm System (NPGS) Ames, Iowa, U.S.A. These two institutions main-
tain the two largest world collections of sunflower. The VIR collection has
about 2,811 accessions, including 493 accessions of wild species and 2,318 of
cultivated origin, most of them collected in the former USSR (Omelchenco
2001). The NPGS sunflower collection maintained at the North Central Regio-
nal Plant Introduction Station (NCRPIS) in Ames contains 3,860 accessions
from 59 countries. The collection includes 1,670 accessions of cultivated
H. annuus, 1,006 accessions of wild H. annuus forms, 430 accessions from
other 11 wild annual Helianthus species, and 754 accessions representing 37
perennial Helianthus species (Marek et al. 2004). It is currently the largest and
most genetically diverse ex situ sunflower collection of the world. NCRPIS not
only conserves this genetically diverse Helianthus collection, but conducts
germplasm-related research, encourages the use of germplasm and associated
information for crop improvement, and distributes the accessions all around
the world.
Other large collections of wild and cultivated sunflower are maintained at the
Institute of Field and Vegetable Crops, Novi Sad, Serbia, at the Dobroudja
Agricultural Institute (DAI) at General Toshevo, Bulgaria, at the Research
Institute for Cereals and Industrial Crops in Fundulea, Romania, and at the
Station d́ Amélioration des Plantes in Montpellier, France.
6.4.1.2 Preservation of In Situ Resources

Preservation of in situ resources (wild species and landraces) in their natural
habitats is critical, especially for wild sunflower populations, because of the lack
of resources necessary to preserve all the wild species in seed banks. Moreover, a
significant proportion of the wild diversity is likely to be lost while regenerating
banked germplasm accessions. Unfortunately, the long-term preservation of
some wild sunflower populations in their natural habitats is not always promis-
ing and some species are endangered or even extinct (Seiler and Rieseberg 1997).
The U.S. Department of Interior, Fish and Wild Life Service listed several
endangered and threatened species (Seiler and Rieseberg 1997). These species
6 Sunflower 161
included the annuals H. paradoxus and perennials H. eggertii and H. schwei-

nitzii. Other candidate species for federal protection were the annuals
H. anomalus, H. deserticola, H. exilis and H. niveus subsp. tephrodes as well as
the perennials H. laevigatus, H. carnosus, H. smithii and H. verticillatus.
As for other genera, the primary obstacle for long-term preservation of wild
Helianthus populations is human activity. This is for example the case of
H. exilis, restricted to serpentine soils in the Inner Coastal Range of California,
where mining activities destroyed several populations (Seiler and Rieseberg
1997). Another example of the impact of human activities was the case of
H. nuttallii subsp. parishii, whose populations were drastically reduced by
urbanizations around Los Angeles in Southern California (Rogers et al.
1982). In addition to the direct destruction of wild populations by development,
their disturbance by human activity favours their hybridization with more
widespread species (Rieseberg 1991). The resulting hybrid plants usually have
lower fitness than locally adapted populations. For example, several rare
annual sunflowers such as H. anomalus, H. paradoxus and H. deserticola
occur sympatrically and occasionally hybridize with the common sunflower,
H. annuus, what might imply a threat for the existence of these species. Another
threat for the preservation of some rare species with small population sizes
(e.g. H. paradoxus and H. deserticola) is their low level of genetic diversity
(Rieseberg 1991).
6.4.1.3 Core Collections

Large germplasm collections usually contain duplications and they are difficult
to manage in the evaluation of the existing variability for useful traits. Accord-
ingly, the establishment of core subsets of the sunflower collections is impera-
tive. Of the 1,624 cultivated accessions of the U.S. NPGS sunflower collection, a
core collection of 112 accessions (7%) was established based on 20 descriptors
(Brothers and Miller 1999). The accessions in this core collection represented 38
of the 57 countries of origin of the whole sunflower collection and contained
2 ornamental accessions, 7 breeding lines, 12 landraces, and 91 cultivars.
6.4.1.4 Genetic Stock Collections

Genetic stocks comprise unique mutants of different traits (morphological,
chemical, physiological) as well as lines with specific characteristics (male-sterile
lines, isolines, aneuploid lines) that are useful for basic research. There are
about 30 sunflower genetic stocks registered by the Crop Science Society of
America. Examples of these genetic stocks are characterised by nuclear male
sterility (Jan 1992a), tetraploidy (Jan 1992b), dwarfness (Velasco et al. 2003a),
altered seed oil fatty acid profile (Miller and Vick 2002; Vick et al. 2007), or
resistance to herbicides (Miller and Al-Khatib 2002).
6.4.2 Germplasm Evaluation
A first step in any breeding program is the identification of genetic variability

for different target traits. Sunflower breeders have been using a wide range of
germplasm of wild and cultivated sunflower to search for variation for agro-
nomic and seed quality traits as well as resistance to insects and diseases.
6.4.2.1 Agronomic and Physiological Traits

World collections of wild and cultivated germplasm possess a wide varia-
bility for morphological and physiological traits (plant height, flowering
period, leaf and achene characteristics, etc.) of interest for sunflower
breeding. A wide variety of agronomic traits was examined in wild
Helianthus species for potential use in improving the hardiness and pro-
ductivity of cultivated sunflower (Laferriere 1986). Wild Helianthus spe-
cies have also been evaluated for resistance to several environmental
stresses. Blanchet and Gelfi (1980) tested 10 species for various aspects
of drought resistance and recommended H. argophyllus as a most likely
source because of its pubescent leaves that reflect sunlight, reduced water
loss, low stomatal resistance, and low transpiration rates. This species has
been frequently used in breeding programs for drought resistance (Blan-
chet and Gelfi 1980). High variability has also been found in wild species
for traits related to photosynthetic efficiency such as leaf area duration
(Škorić 1988).
Several species of Helianthus are native to salt-impacted habitats. For exam-
ple, H. paradoxus is found in saline marshes, where it exhibits high salt tolerance
attributed to great leaf succulence and leaf sodium sequestration (Lexer et al.
2003b). Variability for salt tolerance has been also identified in germplasm of
cultivated sunflower (Ashraf and Tufail 1995).
6.4.2.2 Cytoplasmic Male Sterility

Cytoplasmic male sterility (CMS) is a maternally inherited trait preventing
plants from producing normal pollen. CMS is used as a tool to generate F1
hybrid seed. Based on its origin, CMS is classified as autoplasmic or alloplas-
mic. Autoplasmic CMS refers to the cases where CMS has arisen within the
species as a result of mutational changes in the cytoplasm. Alloplasmic male
sterility arises from interspecific, intergeneric and occasionally intraspecific
crosses due to incompatibility between nucleus and cytoplasm. Both types of
CMS have been identified in sunflower. A type of stable alloplasmic male
sterility named PET1 was reported by Leclercq (1969) in the progeny of an
interspecific cross between H. petiolaris and cultivated sunflower. The subse-
quent identification of dominant fertility restoration genes, especially a source
6 Sunflower 163
derived from wild species (Kinman 1970) allowed for efficient and economical
production of commercial hybrid seed 30 years ago. Virtually all cultivated
sunflower hybrids are currently based on the CMS source derived by Leclercq
(1969). The identification of additional CMS sources has been an important
objective to broaden genetic diversity in cultivated sunflower. As a result of
these efforts, a total of 70 CMS sources were identified (Table 6.3). Most of
these sources originated from progenies of crosses between 16 different wild
Helianthus accessions (mostly annuals) and cultivated lines (Serieys 2002), but
some of them arose from mutational changes within the species H. annuus.
Fertility restoration genes, found primarily in wild Helianthus species and also
in related genera, have been reported for 34 CMS sources. Detailed inheri-
tance studies have been conducted for 19 of the CMS sources (Serieys 2002).
Therefore several CMS systems other than the widely used PET1 are available
for practical hybrid seed production.
Table 6.3 Sources of cytoplasmic male sterility in sunflowera

FAO FAO
Name Origin (species) codeb Name Origin (species) codeb
Kouban H. annuus ANLl HA89 H. annuus MUT 10
lenticularis
Indiana 1 H. annuus ANL2 HA89 H. annuus MUT 11
lenticularis
Vir 126 H. annuus, ANL3 HA89 H. annuus MUT12
lenticularis
397 H. annuus wild ANN1 Anomalus H. anomalus ANOl
517 H. annuus wild ANN2 Argophyllus H. argophyllus ARG1
Ns-Ann-81 H. annuus wild ANN5 Arg3-Ml H. argophyllus ARG3-
M1
Ns-Ann-2 H. annuus wild ANN6 Argophyllus H. argophyllus ARG4
H. annuus wild ANN7 Bolanderi H. bolanderi BOL1
H. annuus wild ANN8 Dv-10 H. debilis DEB1
H. annuus wild ANN9 Exilis H. exilis EXI1
Fundulea 1 H. annuus AMT1 Exi2 H. exilis EXI2
texanus
An-67 H. annuus ANN10 Cmg2 H. giganteus GIG1
An-58 H. annuus ANN11 Cmg3 H. maximiliani MAX1
An-2-91 H. annuus ANN12 H. maximiliani MAX2
An-2-92 H. annuus ANN13 Mollis H. mollis MOL1
H. annuus ANN14 Neglectus H. neglectus NEG1
Cms-G H. annuus ANN15 Canescens H. niveus NIC1
canescens
Cms-Dp H. annuus ANN16 Fallax H. petiolaris PEF1
fallax
Cms-Vl H. annuus ANN17 Pet/Pet H. petiolaris PEP1
petiolaris

FAO FAO
Name Origin (species) codeb Name Origin (species) codeb
H. annuus ANN18 Classical H. petiolaris PET1
Cms Nutt
H. annuus ANN19 Cmg1 H. petiolaris PET2
Nutt
H. annuus ANN20 Petiolaris H. petiolaris PET3
Bis Nutt
H. annuus ANN21 Pet34 H. petiolaris PET4
H. annuus ANN22 H. petiolaris PET5
Hemus H. annuus MUT1 Praecox H. praecox PRA1
Peredovick H. annuus MUT2 Phir-27 H. praecox hirtus PRH1
Stadion H. annuus MUT3 Praecox H. praecox PRP1
praecox
Peredovick H. annuus MUT4 Ppr-28 H. praecox PRP2
praecox
Peredovick H. annuus MUT5 Run-29 H. praecox PRR1
Voronejskii H. annuus MUT6 Resinosus H. resinosus RES1
243
HA89 H. annuus MUT7 Vulpe H. rigidus RIG1
HA89 H. annuus MUT8 Rig-M-28 H. rigidus RIG2
HA89 H. annuus MUT9 Strumosus H. strumosus STR1
a
Serieys (2002).
b
The coding system for CMS sources consists of three-letter abbreviations of the cytoplasm
donor species or subspecies followed by a number starting with 1, depending on the time of
discovery and its reaction to restoration testers.
6.4.2.3 Disease and Insect Resistance

Diseases and insects are limiting factors of production in the majority of sun-
flower producing countries. The most serious diseases of sunflower are caused
by fungi. They include Sclerotinia wilt, stalk rot, and head rot (Sclerotinia
sclerotiorum), Verticillium wilt (Verticillium dahliae), sunflower rust (Puccinia
helianthi), Phoma black stem (Phoma macdonaldii), downy mildew (Plasmopara
halstedii), Phomopsis stem canker (Diaporthe helianthi), charcoal rot (Macro-
phomina phaseolina), alternaria diseases (Alternaria spp.), powdery mildew
(Erysiphe cichoracearum, Sphaerotheca fuliginea, Leveillula tarucia), and
Rhizopus head rot (Rhizopus spp.). Resistance to most of these diseases is
found in wild Helianthus species as shown in Table 6.4 for some examples.
Genetic resistance to broomrape (Orobanche cumana), a parasitic plant that
limited early sunflower production in the former USSR, was initially introduced
into susceptible sunflower mainly from the wild species H. tuberosus (Pustovoit
and Gubin 1974). More recently, results of evaluation of sunflower germplasm
for resistance to new virulent races have shown that wild Helianthus species
constitute the major source of resistance genes, although resistance was also
found in accessions of cultivated material (Fernández-Martı́nez et al. 2000).
6 Sunflower 165
Table 6.4 Reported resistance to important diseases in wild sunflower species

Disease Species with resistance Reference
Downy mildew (Plasmopara H. argophyllus Hoes et al. (1973)
helianthi) H. annuus
H. petiolaris
H. praecox
Verticilium wilt (Vericillium H. annuus Hoes et al. (1973)
dahliae) H. petiolaris
H. praecox
Rust (Puccinia helianthi) H. argophyllus Hoes et al. (1973)
H. annuus Quresh et al. (1993)
H. petiolaris
H. praecox
Alternaria leaf spot (Alternaria H. hirsutus Morris et al. (1983)
helianthi) H. pauciflorus
H. tuberosus
Powdery mildew (Erysiphe H. debilis subsp. Saliman et al. (1982)
cochoracearum) debilis Jan and Chandler (1985)
H. bolanderi
H. praecox
Phoma black stem (Phoma H. decapetalus Škorić (1985)
macdonaldii) H. eggertii
H. hirsutus
H. resinosus
H. tuberosus
Phomopsis stem canker (Diaporthe H. maximiliani Škorić (1985)
helianthi) H. pauciflorus Dozet (1990)
H. hirsutus
H. resinosus
H. mollis
H. tuberosus
Rizopus head rot (Rhizopus H. divaricatus Yang et al. (1980)
arrhizus) H. hirsutus
H. resinosus
H. x laetiflorus
Sclerotinia head rot (Sclerotinia H. decapetalus Pustovoit and Gubin (1974)
sclerotiorum) H. grosseerratus Mondolot-Cosson and
H. nuttallii Andary (1994)
H. pauciflorus Rönicke et al. (2004)
H. resinosus
H. tuberosus
Sclerotinia root rot H. mollis Škorić (1987)
(Sclerotinia sclerotiorum) H. nuttallii
H. resinosus
H. tuberosus
Sclerotinia mid-stalk (Sclerotinia H. praecox Škorić (1987)
sclerotiorum) H. giganteus
H. maximiliani
H. pauciflorus
H. resinosus
H. tuberosus
Broomrape (Orobanche cumana) Most of the perennial Fernández-Martı́nez et al.
species (2000)
Although several hundreds of insect species are associated with sunflower,

only a few of them are economically important pests of cultivated sunflower
(Schulz 1978). An exception was the significant yield reduction caused by the
European sunflower moth (Homoeosoma nebulellum, Lepidoptera) in the
former USSR at the end of the 19th century, which encouraged the first
scientific breeding research on sunflower as early as 1890. Resistant cultivars
were developed by interspecific hybridization of cultivated sunflower with H.
tuberosus, which accumulates phytomelanin in the seed, thus reducing larval
feeding (Gundaev 1971). Some wild species have been found to be resistant to
insect pests attacking sunflower in North America, such as the sunflower stem
weevil (Smicronyx fulvus, Coleoptera) (Rogers and Seiler 1985) and the
sunflower beetle (Zygogramma exclamationis, Coleoptera) (Rogers and
Thompson 1980). Evaluation of germplasm of cultivated sunflower has also
revealed the existence of variability for resistance to several important insect
pests (Charlet et al. 2007).
6.4.2.4 Oil and Protein Content and Quality

Oil and protein content of the sunflower achene depends on both the percentage
of hull and the oil and protein concentration in the kernel. Variation for hull
percentage and oil and protein content has been found in extensive evaluations
of cultivated sunflower germplasm (Jiménez et al. 1985; Miller et al. 1992).
Variation for oil and protein content also exists in wild species. Maximum oil
content reported in wild species is lower than current standards of cultivated
sunflower (around 50%). Conversely, maximum values of protein content
found in wild species (35–40%) are generally higher than the typical protein
content of cultivated sunflower (Seiler 1984; Ruso et al. 2000).
Variation for the seed oil quality components has been also found through
the evaluation of genetic resources. The evaluation of germplasm collections led
to the identification of cultivated sunflower germplasm with reduced levels of
saturated fatty acids (Vick et al. 2002), high palmitic acid content (Demurin
2003) or high linoleic acid content (Miller and Vick 2001), as well as wild
sunflower germplasm with reduced levels of saturated fatty acids species (Seiler
2004). Variation for increased levels of beta- and gamma-tocopherol has been
found in collections of cultivated germplasm (Demurin 1993; Velasco et al.
2004a).
Sunflower germplasm is also a useful source of variation for reducing
antinutritive compounds of the seeds, such as chlorogenic acid, that reduces
the nutritive value of the meal. Dorrell (1976) found variation for reduced
levels of chlorogenic acid in germplasm of wild and cultivated Helianthus
species.
6 Sunflower 167
6.5.1 Development of High Oil Germplasm in the Former USSR
After its introduction to Europe in the 16th century, sunflower was mainly
grown as an ornamental. The first mention of sunflower cultivation as an oil
crop was in Russia in 1779 (Gundaev 1971). The crop expanded rapidly and the
first local varieties were developed by the end of the 19th century. These
varieties were selected in small garden plots under different environmental
conditions, which led to a wide range of variation for different traits such
a maturity and seed types, including well-filled, round seeds with thin hull
and oil content of about 20–30%, used for oil extraction, and large, long
seeds with thick hull and oil content about 15–20%, used for direct human
consumption (Gundaev 1971). Moreover, there was an important local selec-
tion for resistance to the European sunflower moth (Homoeosoma nebulella)
and sunflower broomrape (Orobanche cumana), which at that time jeopardized
the survival of the crop.
Scientific sunflower breeding started in 1910–1912 at Krasnodar by the
academician V.S. Pustovoit, based on the local varieties developed at a local
scale during the previous century (Panchenco 1966). The main efforts of
breeders were initially devoted to the control of broomrape and sunflower
moth, but the development of varieties with high oil content by V.S. Pustovoit
constituted a crucial milestone in the development of sunflower as an oil crop
not only in the USSR, but also throughout the world. The local varieties
cultivated in Russia in 1913 contained only 30–33% of oil in dry seeds. This
percentage increased up to 43% in 1935, 46% in 1953 and 51% in 1958, when
the variety ‘‘Peredovik’’ was released (Panchenco 1966). This progress was
obtained through the use of Pustovoit’s ‘‘Method of Reserves’’, a method of
individual selection with progeny testing and controlled pollination (Pusto-
voit 1967), together with the use of accurate laboratory techniques for oil
content analysis. Moreover, this spectacular increase of oil content of the
achenes did not caused any decline in the seed yield of the varieties released.
The open pollinated Russian cultivar ‘‘Peredovik’’, with high oil content,
introduced during the 1960s in the western countries (US, Canada, Western
Europe), was the base of the first sustained commercial production of oilseed
sunflower in these countries (Fick and Miller 1997).
6.5.2 Utilization of the Inbred-Hybrid Method

One of the most important breeding milestones in sunflower has been the
development of hybrid cultivars that made possible the utilization of heterosis.
Initial studies in the former USSR (Morozov 1947) and Canada (Unrau 1947;
Putt 1962) indicated that experimental hybrids outyielded check varieties from
160 to 189%. However, the practical production of hybrid seed was hindered by
the absence of a suitable type of male sterility.
The first commercial sunflower hybrids were produced in Canada during the
1950s using a female parent with a high degree of self-incompatibility and
allowing cross pollination with appropriate male parents. Although this
method resulted in seed lots with hybridization rates as high as 90% under
favourable environments, in general the percentage of hybrid seed was usually
below 50% (Putt 1962). Thus, the seed produced by the self-incompatibility
system often did not meet the legal requirements for designation as hybrid seed.
The existence of nuclear male sterility was first reported in the former USSR
(Kuptok 1935) and later in France (Leclercq 1966) and Canada (Putt and
Heiser 1966). In most cases the trait was controlled by a single recessive gene.
Nuclear male sterility was used to produce hybrid seed in France and Romania
during the early 1970s. The identification of a close linkage between genes for
male sterility and anthocyanin pigmentation (Leclercq 1966) facilitated
the identification and removal of the male fertile plants prior to flowering,
thus allowing nearly 100% hybridization. This system allowed the development
of the first commercial hybrids in Romania and in France, which yielded up to
24% more than the open pollinated varieties (Vrânceanu 1974). Even though
the nuclear male sterility was an important step in the development of hybrid
sunflowers, it required a considerable manual labour to remove male
fertile plants.
The discovery of cytoplasmic male sterility, with its inherent advantages,
provided a highly efficient method for commercial production of hybrid seed.
The first stable source of cytoplasmic male sterility was discovered by Leclercq
in 1968 from an interspecific cross involving H. petiolaris and H. annuus
(Leclercq 1969). Subsequent identification of genes for fertility restoration
in wild species (Kinman 1970) and in certain obsolete sunflower cultivars
(Vrânceanu and Stoenescu 1971) allowed the efficient and economical pro-
duction of hybrid seed. The development of the first sunflower hybrids based
on cytoplasmic male sterility in the early 1970s intensified the interest of seed
companies on the crop, which led to a considerable increase of sunflower
production in many countries. When comparing sunflower yields in the coun-
tries that grew open-pollinated varieties before the introduction of hybrids,
seed yields increases of about 20% were estimated (Fick and Miller 1997).
6.5.3 Development of New Types of Oil
The development of new oil types has been another important achievement
for the sunflower oil industry. Sunflower breeders have been tremendously
successful in developing mutants with new types of oil in the last 30 years,
opening the possibility of tailoring specialty oils for specific market niches
6 Sunflower 169
(Fernández-Martı́nez et al. 2004). The most relevant was the high oleic acid
mutant identified in Russia in the seventies (Soldatov 1976), which has made
possible the development of commercial cultivars with mid oleic acid content
(55–75%) and high oleic acid content (>85%), which are currently cultivated all
over the world. In the U.S., the mid oleic acid varieties, locally known as
Nusun1, are estimated to represent more than 85% of the total oilseed sun-
flower acreage according to the data of the National Sunflower Association.
6.6.1 Seed Yield
One of the basic goals of sunflower breeding is to increase grain yield. The
introduction of hybrid cultivars and the consequent exploitation of heterosis
represented a breakthrough that produced an increase in yield potential around
25%. No significant improvement in grain yield potential has been observed at
large scale before or after this turning point (López-Pereira et al. 1999). Even
though several studies have identified yield components with a direct effect on
seed yield, such as number of grains per head and grain weight (Connor and
Hall 1997), major achievements in improving grain yield in sunflower have been
more related to improving combining ability of hybrid parents or to selection
for adaptation to limiting conditions in specific areas, e.g. shorter plant stature
in areas with great lodging risk (Schneiter 1992), high degree of self-fertility in
areas with limited pollinator populations (Miller et al. 1982), or pronounced
head inclination in areas with high temperature and intense sunlight or with
high risk of bird predation (Hanzel 1992). In many sunflower production areas,
improved performance of recent hybrids was related to increased disease resis-
tance, e.g. Verticillium wilt in Argentina (Sadras et al. 2000), Phomopsis stem
canker in former Yugoslavia (Škorić 1985), or broomrape in several European
countries (Alonso et al. 1996).
6.6.2 Morpho-Physiological Traits
6.6.2.1 Plant Height

The cultivated sunflower is a tall, erect, unbranched plant with a plant height
below 75 cm in dwarf types to more than 5 m in giant varieties. Most common
cultivated hybrids have a stem height of 160–180 cm, although the trait is very
dependent on the environment. Several genetic sources of plant dwarfness have
been identified and both semi-dwarf (100–160 cm) and dwarf (50–100 cm)
hybrids have been produced and compared to standard-height hybrids. In
general, no clear agronomic advantages were associated with reduced plant
height in standard environments (Schneiter 1992; Velasco et al. 2003a). The
major advantage of semidwarf and dwarf cultivars is their resistance to lodging

in environments with risk of heavy rains and strong winds during the growing
season.
Plant height in standard sunflower types is regarded as a quantitative trait
(Lay and Khan 1985). Several types of reduced plant height (<100 cm) have
been developed. Reduced plant height in lines with a reduced number of leaves
has been reported to be controlled by a single recessive gene (Miller and Fick
1997). Reduced plant height in genotypes with reduced internode length and a
standard number of leaves has been found to be quantitatively inherited (Miller
and Hammond 1991), controlled by a single dominant gene (Miller and Fick
1997), or by two recessive genes (Velasco et al. 2003b).
6.6.2.2 Head Size, Shape and Inclination

Head diameter may vary from 6 to 75 cm. The head shape presents a large
gradation from concave to convex, whereas the head angle may vary from 0
(horizontal facing upwards) to 1808 (horizontal facing downwards). The three
traits are quantitatively inherited and subject to environmental effects (Miller
and Fick 1997).
Classical Russian sunflower breeders put special emphasis on the importance
of optimal head size and head shape to maximize sunflower yield. Morozov
(1947) considered medium-size, thin and flat head as the ideotype for sunflower.
Similarly, Pustovoit (1966) considered that a medium-size head (20–25 cm), in
combination with adequate plant density, was one of the main determinants of
grain yield. Larger heads would increase the percentage of hull as well as the
number of empty grains in the center of the head.
Specific head shape and head inclination types have been identified as
advantageous under certain environments. The incidence and severity of certain
diseases such as white rot caused by Sclerotinia sclerotiorum and gray rot caused
by Botrytis cinerea is directly related to the angle of the head. The lowest disease
incidence is observed when the head is at an angle of 458 and remains above the
foliage (Škorić 1992). A pronounced head inclination around 1808, in which
the head is parallel to the soil surface, is desired to prevent sun scald in areas
with high temperature and intense sunlight during seed maturation (Sailsbery
and Knowles 1983), as well as to reduce bird predation in combination with a
concave-shaped head (Hanzel 1992).
6.6.2.3 Flowering and Maturity Dates

Cultivation of sunflower varieties with flowering and maturity dates adapted to
the particular agroecological conditions of a region is essential to ensure a high
productivity of the crop. Most sunflower cultivars exhibit quantitative long-day
or day-neutral responses to photoperiod although there are differences in
photoperiodic sensitivity of sunflower genotypes (Connor and Hall 1997).
A great variation in days from planting to maturity can be found in sunflower,
6 Sunflower 171
from around 75 to 150 days (Fick 1978). The genetic control of flowering date is
rather complex and contradictory results have been obtained depending on the
germplasm used. Vrânceanu (1974) suggested that the number of days to
flowering was controlled by many genes, some of them affecting photoperiod-
ism. However, most studies have reported a high heritability of flowering date
(Miller and Fick 1997).
6.6.2.4 Pollen Self-Compatibility and Flower Characteristics

Sunflower inflorescence is a capitulum, usually referred to as head, formed by
an outer whorl of sterile, ligulate flowers, known as ray flowers, and a varying
number of inner whorls arranged in spiral from the head centre containing
fertile flowers, known as disk flowers or florets. Flower nectaries are located
at the base of the style in disk flowers and they play an important role in
attracting pollinators (Tepedino and Parker 1982). Wild sunflowers have a
system of sporophytic self-incompatibility that promotes insect-mediated
cross-pollination. The number of loci involved in self-incompatibility has
been disputed. One multiallelic locus was identified by Fernández-Martı́nez
and Knowles (1978). Self-incompatibility systems were maintained in open-
pollinated populations, but hybrid breeding has been accompanied by intense
selection for high levels of self-compatibility. Nowadays, most commercial
hybrids are virtually self-fertile (Fick and Miller 1997). Nevertheless, pollinator
(mainly bees) attractiveness is still important in fields of hybrid seed production,
where the production success depends on efficient pollen transfer from male-
fertile to male-sterile parents. Selection for bee attractiveness is complex, since
nearly every flower trait influences attractiveness to bees. Some of the most
important traits are a short corolla length, short styles, unpigmented stigmas,
and total sugar content and profile in the nectar (Montilla et al. 1988).
6.6.2.5 Male Sterility

The availability of male sterility is essential for the commercial production of
hybrid seed. Both nuclear male sterility (NMS) and cytoplasmic male sterility
(CMS) have been identified in sunflower. Nuclear male sterility is generally
controlled by a recessive gene. Eleven different genes named Ms1 through Ms11
have been identified in NMS lines (Jan 1992c). A total of 72 unique CMS
sources have been identified (Table 3; Serieys 2002), fertility restoration genes
are available for 34 of them. Most of the CMS sources require at least two genes
for fertility restoration, although some of them show single-gene restoration
(Jan and Vick 2007).
6.6.2.6 Oil, Protein and Fibre Contents

The fruit of sunflower is an achene, commonly known as sunflower seed or
grain. The kernel to hull ratio is one of the main parameters defining the
profitability of the crop. The percentage of hull in the achenes is very variable in
sunflower germplasm, from around 10 to 60% (Miller and Fick 1997). In
current oilseed cultivars, the kernel represents more than 75% of the total
achene weight.
More than 80% of the economic value of oilseed sunflower cultivars is
obtained from the extracted oil, whereas the rest is obtained from the protein-
rich meal that remains after oil extraction. One of the major breeding
achievements that facilitated the expansion of sunflower as one of the most
important world oilseed crops was a drastic increase of oil content of sun-
flower achenes, from around 30% to more than 50% (Panchenco 1966).
According to Alexander (1963), two thirds of the increase in oil content was
produced by a reduction of the hull percentage in the achenes, whereas one
third was produced by the increase of oil content in the kernel. The increment
in the kernel to hull ratio was also associated with a concomitant increase of
protein content, around 17% in current cultivars, and a reduction of fibre
content. Additionally, the industry uses to hull the achenes before crushing to
reduce the fibre content and improve the digestibility of the meal. Accord-
ingly, the ease of hulling is also a selection target to take into account (Denis
et al. 1994).
The hull content is a quantitative trait with high heritability mainly governed
by genes with additive effects (Kovacik and Skaloud 1990). The ease of hulling
is also a trait with high heritability for which variation in sunflower germplasm
has been identified (Denis et al. 1994). Both oil and protein contents in sun-
flower achenes are traits quantitatively controlled by the genotype of the plant
(sporophytic control) (Pawlowski 1964), with predominance of additive gene
effects and medium to high heritabilities (Fick 1975; Alza and Fernández-
Martı́nez 1997).
6.6.2.7 Oil Quality

Sunflower oil mainly contains molecules of triacylglycerol, composed of three
fatty acids attached to a glycerol skeleton, which represent more than 95% of the
total oil weight. The rest are lipid and lipid-soluble compounds, some of them of
great value because of the functional and nutritional properties that confer to the
oil. Breeding for oil quality in sunflower has been mainly focused in the mod-
ification of the fatty acid profile of triacylglycerols, although minor compounds
with important nutritional and antioxidant value such as tocopherols and phy-
tosterols have also attracted the attention of plant breeders in recent years.
The seed oil of conventional sunflower varieties is characterised by a high
proportion of the unsaturated oleic acid (18:1) and linoleic acid (18:2), which
together account for about 90% of the total fatty acids (Table 6.5). The
remaining 10% correspond to the saturated palmitic acid (16:0) and stearic
acid (18:0). The relative proportion of oleic acid and linoleic acid is strongly
influenced by the environmental conditions, especially temperature, during
seed development (Harris et al. 1978).
6 Sunflower 173
Table 6.5 Outstanding sunflower germplasm producing seed oil types with modified fatty
acid and tocopherol profiles
Fatty acid compositiona (%)
Oil type 16:0b 18:0 18:1 18:2 Reference
Standardc 7 3 30 60
Low satd 4 3 40 52 Vick et al. (2002)
High 16:0 25 4 11 55 Osorio et al. (1995)
High 18:0 5 26 14 55 Osorio et al. (1995)
Mid 18:1 4 5 55 34 Vick and Miller (1996)
High 18:1 5 3 90 2 Soldatov (1976)e
Tocopherol composition (%)
Oil type a-T b-T g-T d-T Reference
Standard 95 4 1 0
Medium b-T 50 50 0 0 Demurin (1993)
High b-T 25 75 0 0 Velasco et al. (2004b)
High g-T 5 0 95 0 Demurin (1993)
High d-T 5 0 30 65 Velasco et al. (2004b)
a
Percentages for the four major fatty acids are given only.
b
16:0=palmitic acid, 18:0=stearic acid, 18:l=oleic acid, 18:2=linoleic acid.
c
Averaged from cold and warm environments.
d
Low content of total saturated fatty acids.
e
Data of hybrids developed by Fernández-Martı́nez et al. (1993) from a mutant originally
developed by Soldatov (1976).
The tocopherols are a group of four lipid-soluble substances with molecular

structure comprised of a chromanol ring and a saturated phytyl side chain.
The four tocopherols, named alpha-, beta-, gamma-, and delta-tocopherol
differ in the number of methyl substituents and the pattern of substitution in
the chromanol ring (Packer and Obermüller-Jevic 2002). They exhibit differ-
ential in vivo and in vitro antioxidant activities. While alpha-tocopherol exerts
a maximum in vivo activity, also known as vitamin E activity, but poor in vitro
protection of the extracted oil, gamma-, delta- and to a lesser extent beta-
tocopherol are powerful in vitro antioxidants with low vitamin E value (Pon-
gracz et al. 1995). However, the tocopherol profile is only a part of the picture
when designing a breeding programme aimed at improving the tocopherol
fraction in sunflower seeds. The other part is the total tocopherol content.
The seed oil of conventional sunflower varieties has an average total tocopherol
content of 708 mg kg–1, mainly in the alpha-tocopherol form, which accounts
for more than 90% of the total tocopherols (Padley et al. 1994).
Phytosterols or plant sterols are essential components of the cell membranes.
Their role as functional food components and nutraceuticals due to their ability
to lower total and LDL serum cholesterol in humans is increasing in recent
years. Chemically, they are steroid alcohols (triterpenes) synthesized from
squalene in the isoprenoid pathway (Piironen et al. 2000). Vegetable oils are
the richest natural sources of plant sterols. The seed oil of conventional sun-
flower varieties has an average sterol content of 3,387 mg kg–1, mainly as
sitosterol (59.9% of the total sterols), D7 stigmastenol (10.4%), campesterol

(9.5%), and stigmasterol (9.5%) (Padley et al. 1994).
The optimal quality of sunflower oil depends on the intended use of the oil,
either for food or non-food applications. The former include salad and cooking
oils as well as oils for the food industry (margarines, shortenings, etc.).
The latter comprises countless industrial sectors such as biofuels, lubricants,
surfactants, surface coatings, cosmetics, plastics, etc. In general, those oil
characteristics that are undesirable for a particular application are required
for others. A clear example is the saturated fatty acid content in sunflower oil.
Saturated fatty acids are regarded as detrimental on human health because of
their contribution to raise cholesterol levels as compared with isocaloric
amounts of carbohydrates (Mensink et al. 1994). Accordingly, the breeding
objective to produce a healthy oil of direct consumption (e.g. salad oil) is to
reduce total saturated fatty acid content. But on the other hand, a sunflower oil
rich in saturated fatty acids is desirable for the industry of margarines and
related products, because its semi-solid consistency reduces the need for trans-
formations such as hydrogenation or transesterification that generate trans and
positional isomers related to heart disease (Willett and Ascherio 1994). Accord-
ingly, the development of sunflower germplasm producing oil rich in saturated
fatty acids is also a breeding objective if the oil is intended to the production of
semisolid fats.
Oleic acid (18:1, n-9) is nowadays considered as the preferred fatty acid for
edible purposes, as it combines a hypocholesterolemic effect (Mensink and
Katan 1989) with a much greater oxidative stability than polyunsaturated
fatty acids. For this reason, selection for mid and high oleic acid contents has
been a priority in sunflower (Table 6.5).
The tocopherols are another good example of contrasting breeding goals
depending on the intended use of the oil. Selection for high alpha-tocopherol
content would enhance the vitamin E value of the oil in human nutrition, but
selection for low alpha tocopherol content would result in the accumulation of
tocopherols with greater in vitro antioxidant effect and consequently in optimal
oil properties for applications requiring high oxidative stability, e.g. deep frying
or biolubricants.
A summary of outstanding sunflower germplasm producing seed oil types
with modified fatty acid and tocopherol profiles is presented in Table 6.5.
In general, genetic modifications altering either the fatty acid or the tocopherol
profile have been found to be qualitative rather than quantitative, i.e. they are
controlled by a reduced number of genes and they are less affected by environ-
mental factors than quantitative traits such as oil content (Fernández-Martı́nez
et al. 2004). Conversely, total tocopherol content in sunflower seeds is regarded
as an oligogenic or polygenic trait, although no genetic studies on this trait have
been conducted yet. Preliminary information suggests an important contribu-
tion of the genotype to the expression of the character (Alpaslan and Gündüz
2000; Velasco et al. 2002).
6 Sunflower 175
The high oleic acid content in sunflower was initially identified as a mono-
genic trait produced by dominant alleles Ol (Urie 1984), although more detailed
studies identified several modifying genes affecting the Ol gene and producing
reversal of the expected dominance (Miller et al. 1987; Fernández-Martı́nez
et al. 1989b; Pérez-Vich et al. 2002b), which has complicated the practical
management of the trait in breeding programmes. High palmitic acid content
has been found to be controlled by alleles at three independent loci P1, P2, and
P3 in such a way that the high palmitic acid phenotype is expressed in genotypes
that are homozygous recessive at the P1 locus and either at P2 or P3 (Pérez-
Vich et al. 1999a). Genetic characterization of high stearic acid content of the
sunflower mutant CAS-3 concluded that the trait is controlled by partially-
recessive alleles at two loci Es1 and Es2 (Pérez-Vich et al. 1999b). A third gene
involved in high stearic acid content, Es3 was identified in the mutant CAS-14.
However, genetic recombination of es3 alleles with es1 and es2 alleles from
CAS-3 did not result in an increment of the maximum stearic acid content in the
seeds compared to the maximum levels produced by the es3 alleles alone (Pérez-
Vich et al. 2006a). Reduced saturated fatty acid content was identified as a
partially dominant trait controlled by more than one gene (Vick et al. 2002).
The genetic studies conducted so far have also revealed that the modified
tocopherol profiles in sunflower seeds are also controlled by recessive alleles at a
reduced number of loci. Demurin et al. (1996) found that recessive alleles at the
Tph1 locus produced mid beta-tocopherol levels, recessive alleles at the Tph2
locus produced high gamma-tocopherol content, whereas their genetic recom-
bination resulted in increased delta-tocopherol content. Hass et al. (2006)
identified a third gene, named Tph3, which in combination with Tph1 and
Tph2 produced a high delta-tocopherol content.
An important feature of the genetic control of the seed oil quality traits is,
that in most cases they are determined by the genotype of the developing
embryo with small or no maternal influence. The latter is crucial in breeding
programs, as a low weight of maternal inheritance allows selection to be carried
out at a single-seed level. It is also noteworthy that most of the genetic mod-
ifications are recessive, which determines that the modified alleles have to be
introgressed in both parents in hybrid seed production (Fernández-Martı́nez
et al. 2004).
6.6.2.8 Seed Meal Quality

Sunflower seed meal is extensively used as a protein supplement in animal feed
because of its high protein content of around 40% if the achenes are hulled
before oil extraction or around 28% if no hulling occurs (Dorrell and Vick
1997). The principal factors defining sunflower meal quality are a low fibre
content, high protein content of good quality, and absence or low presence of
minor components with detrimental properties from the nutritional or techno-
logical points of view.
Fibre is a heterogeneous chemical entity that includes those carbohydrates

that are not truly digested by the animal and therefore do not contribute energy
when consumed. High fibre content in the meal reduces its nutritive value and
digestibility. It is predominantly associated with the seed hull. Because of the
high percentage of hull in sunflower achenes compared to other oilseeds, hulling
is a common practice necessary to render a meal with adequate quality for
animal feed (Bell 1989).
Sunflower seed meal has a balanced amino acid composition except for
lysine, for which it is deficient (Norton 1989). Accordingly, selection to increase
lysine content in sunflower seeds is one of the major objectives to improve seed
meal nutritive quality. Several studies have reported the existence of variability
for lysine content in germplasm of cultivated (Ivanov 1975) and wild sunflower
accessions (Christov et al. 1993). Even though selection efforts have been
scarce, Ivanov (1975) reported good response to selection for this trait.
Sunflower seeds contain a series of minor compounds that remain in the meal
after oil extraction and confer negative properties from the nutritional and/or
technological point of view. One of these compounds is phytic acid (myoinositol
1, 2, 3, 4, 5, 6-hexakis-dihydrogen phosphate), which is present in cereals and
oilseeds. Sunflower kernels contain around 2.2% phytic acid, which determines
a content around 4.5% in the seed meal (Miller et al. 1986). Phytic acid is a
strong chelating agent that can bind metal ions, reducing the availability of
calcium, iron, magnesium, zinc and other trace elements (Oberleas et al. 1966).
Additionally, phytates form complexes with amino acids, reducing the value of
sunflower meal for nonruminant livestock (Erdman 1979).
Sunflower seeds contain significant amounts of phenolic compounds, mainly
chlorogenic acid and caffeic acid, which reduce the nutritive value of sunflower
meal for animal feed by interacting with amino acids, denaturing proteins, and
inhibiting enzymes (Sozulski 1979). Additionally, chlorogenic acid produces a
yellow-green coloration following oxidation in sunflower meal, which repre-
sents a serious limitation for the use of sunflower meal for human consumption
in form of sunflower flour, protein concentrates and protein isolates (Dorrell
and Vick 1997). Since phenolic compounds are predominantly present in the
seed kernels, hulling scarcely reduces their presence in the meal (Pedrosa et al.
2000). From the evaluation of several lines with contrasting levels of chloro-
genic acid in several environments and evaluation of progenies derived from
crosses involving them, Dorrell (1974) concluded a significant effect of the
genotype on the expression of the trait.
6.6.2.9 Disease Resistance

Most of the pathogens affecting sunflower have only economic impact at a local
scale or under specific environmental conditions, but some of them have great
relevance and can produce important yield losses if no adequate control mea-
sures are adopted. Breeding for resistance is considered the most effective and
sustainable means of control.
6 Sunflower 177
Both qualitative or vertical and quantitative or horizontal genetic resistance

mechanisms have been identified in wild sunflower species and successfully
transferred to cultivated strains. Single dominant genes, generally associated
with race-specific resistance to important diseases such as sunflower rust (Jan
et al. 2004; Jan and Gulya 2006a), downy mildew (Miller and Gulya 1988; Jan
and Gulya 2006a) and Verticillium wilt (Radi and Gulya 2007) have been
identified in wild Helianthus germplasm and successfully transferred to culti-
vated sunflower. A serious problem associated with the use of major gene
resistance is the regular appearance of new races of the pathogen that overcome
the existing resistance genes, determining the need for identifying additional
resistance genes to be introgressed into high yielding cultivars. Since resistance
genes are usually identified in germplasm of wild species, the recovery of good
agronomic characteristics after the introgression of the new resistance gene is a
difficult task. Therefore genetic resistance based on more durable strategies
such a combination of both vertical and horizontal resistance mechanisms has
been proposed (Vear 2004). Pyramiding of resistance genes has been also
proposed as a strategy to develop durable resistance. Tourvieille et al. (2004)
compared different methods to enhance durable resistance to downy mildew,
reporting that gene pyramids were less effective in reducing the appearance of
new races compared to other control methods such as the use of combinations
of resistance Pl genes, by alternation or in ‘‘multi-hybrids’’.
The situation has been different for diseases produced by Sclerotinia scler-
otiorum (wilt, stalk rot, head rot), which cause the greatest losses to sunflower
on a global basis. The causal agent is a polyphagous fungus that attacks many
different species, including sunflower, where infection affects the root, stem,
head, and seeds (Gulya et al. 1997). In this case, resistance is expected to be
complex and controlled polygenically (Jan and Seiler 2007). Genetic resistance
to Phomopsis stem canker (Vrânceanu et al. 1992) and Alternaria leaf blight
(Morris et al. 1983) have been postulated to be oligogenic.
Virus diseases are not currently a major concern in global sunflower
production. More than 30 different viruses have been identified on sunflower,
but only a strain of tobacco streak virus has been reported as a serious disease
of sunflower in all major sunflower-growing regions of India (Ravi et al.
2001). Although resistance to this virus has not been reported yet, resistance
to other viruses such as the sunflower mosaic potyvirus (Jan and Gulya
2006b) and the sunflower chlorotic mottle virus (Lenardon et al. 2005) have
been identified. The latter was found to be controlled by a single dominant
gene.
6.6.2.10 Broomrape Resistance

Sunflower broomrape (Orobanche cumana Wallr.) is a weedy parasitic
angiosperm that represents nowadays a serious constrain for sunflower pro-
duction in Southern Europe and in the Black Sea region. Although the use
of herbicides is being considered as a promising control measure, genetic

resistance offers the most effective and feasible control against O. cumana.
However, the introduction of new resistance has been frequently followed by
the appearance of new pathogenic races overcoming the resistance and there
is a continuous need for new sources of resistance. Vrânceanu et al. (1980)
identified five pathogenic races, named A through E, with a set of sunflower
differentials carrying the dominant resistance genes Or1 through Or5, respec-
tively that provide an accumulative resistance to broomrape races. New
virulent races overcoming resistance gene Or5 have been identified in Spain
(Alonso et al. 1996; Molinero-Ruiz and Melero-Vara 2005), Romania
(Păcureanu et al. 2004), Bulgaria (Shindrova 2006), and Turkey (Kaya
et al. 2004).
Resistance to races A through E has been found in most studies to be under
the control of dominant alleles at single loci (Vrânceanu et al. 1980; Sukno et al.
1999), although two dominant genes (Domı́nguez 1996) and one recessive gene
(Ramaiah 1987) have also been reported. Genetic resistance to Spanish race F
in germplasm derived from wild Helianthus spp. has been found to be controlled
by a single dominant gene Or6 (Pérez-Vich et al. 2002a), which exhibited
incomplete dominance in some environments due to the effect of a second
gene Or7 (Velasco et al. 2006), whereas resistance in germplasm of cultivated
sunflower was controlled by two recessive genes (Akhtouch et al. 2002). A single
dominant gene is involved in the resistance to race F in germplasm developed in
Romania (Păcureanu et al. 2004).
6.6.2.11 Insect Resistance

Insects do not represent a serious threat to sunflower production except in
North America, where insect pests of sunflower coevolved with their native
hosts (Jan and Seiler 2007). Nowadays, the principal strategy in search for
resistance to sunflower infesting insects is the evaluation of wild Helianthus
spp. and cultivated sunflower germplasm.
6.6.2.12 Resistance to Bird Depredation

Bird damage causes serious losses in sunflower production, especially during
the first three weeks of seed maturation (Cummings et al. 1989). Breeding for
some morphological traits contributes to confer resistance to bird predation.
These traits include long involucral bracts, horizontally oriented heads facing
downwards, concave heads, and long head-to-stem distances (Gross and
Hanzel 1991).
6.6.2.13 Resistance to Abiotic Stresses

Sunflower is grown in areas prone to several types of environmental stresses,
from drought to waterlogging, from heat to frost injury, or from poor soils to
6 Sunflower 179
excess salinity. The development of cultivars adapted to such conditions is

critical. But adaptation not only implies the ability to survive under stress,
but the plants must be able to maintain adequate yields.
Several strategies have been followed to select for drought resistance in
sunflower. Avoidance of high evaporative demand periods has been advo-
cated as a strategy to increase productivity in areas prone to drought. In
southern Spain, the shift in sunflower planting dates from spring to winter
resulted in a substantial yield increase (Gimeno et al. 1989). Selection for early
vigour has been proposed as a means for improvement of sunflower yield
under water-limited environments (Agüera et al. 1998). There is also evidence
that improved osmotic adjustment capacity is a trait that contributes to yield
maintenance under drought conditions in sunflower (Chimenti et al. 2002).
Some wild Helianthus spp., especially H. argophyllus, have been suggested as a
source of useful traits to improve water use efficiency such as higher stomatal
densities and leaf pubescence, which reduce transpiration rates (Blanchet and
Gelfi 1980).
6.6.2.14 Herbicide Resistance

The acreage of transgenic varieties of several major crops carrying genes for
resistance to herbicides have been steadily increasing in recent years in many
countries. In sunflower, resistance to imidazolinone and sulfonylurea herbi-
cides was identified in weedy populations of H. annuus (Al-Khatib et al.
1998). The trait has been transferred to cultivated sunflower germplasm
(Miller et al. 2006a). Herbicide resistance in sunflower is mainly controlled
by one gene exhibiting partial dominance and also affected by a second gene
in some genetic backgrounds (Bruniard and Miller 2001). Resistance to
herbicides in sunflower is also being used to control broomrape (Domı́nguez
et al. 2004).
6.6.2.15 Nonoilseed Sunflower

Nonoilseed sunflower seeds are used as snack food, in the confectionery
industry, and for feeding birds and small pets. The seeds of nonoilseed
cultivars have lower seed oil content than oilseed cultivars. Breeding goals
in relation to nonoilseed sunflower largely depend on the requirements of
specific markets. For example large achenes are preferred for uses as snack
food, but small achenes are preferred for birdseed. In some types of soils,
sunflower accumulates excessive concentrations of cadmium in the seed
kernels, which has became a problem for confectionery sunflower. This has
encouraged breeding for reduced cadmium uptake into kernels (Miller et al.
2006b).
6.7.1 Breeding Methods
There are comprehensive reviews of breeding methods and techniques in sun-

flower (Škorić 1988; Miller 1987; Fick and Miller 1997). Four important
features have to be considered in breeding sunflower: (a) obtaining or develop-
ing sources of genetic variability, (b) improving source populations, (c) improv-
ing open pollinated cultivars, and (d) developing lines or parents for producing
hybrid cultivars.
6.7.1.1 Obtaining or Generating Sources of Genetic Variability

The Use of Existing Genetic Variation
Many sources of genetic variability from cultivated germplasm and wild species
are available in germplasm collections to be used in breeding programs. For
example, open-pollinated varieties with high oil content developed in the former
USSR were the base for the initial commercial production of oilseed sunflower
in western countries in the 1960s (Putt 1997). Evaluation of cultivated germ-
plasm has been extensively used to identify useful variation for many different
traits such as disease resistance (Škorić 1992) or oil quality (Demurin 2003).
However, wild species represent the most diverse source of genetic variability in
sunflower breeding. Genes for disease and insect resistance, oil and protein
content and quality, cytoplasmic male sterility, or agronomic and physiological
traits have been identified in wild Helianthus species and transferred into
cultivated lines. The main problem using wild species as sources of variability
is that many of them do not cross readily with the cultivated sunflower.
Transferring genes from wild annual species into cultivated lines can be
accomplished rather easily with conventional crossing and backcrossing. For
example, genes conferring resistance to several diseases such as rust and
downy mildew have been transferred from H. debilis or wild H. annuus to
cultivated sunflower (Quresh et al. 1993; Tan et al. 1992). Conversely, crossing
perennial Helianthus species with the cultivated sunflower is generally difficult
due to early hybrid embryo abortion as well as high levels of sterility in the F1
or BC F1 generations (Jan 1997). These problems can be avoided with the
utilization of the embryo culture technique (Chandler and Beard 1983) and
subsequent chromosome doubling of the F1 (Jan 1988). Using these techni-
ques, amphiploids of the wild species H. gracilentus, H. hirsutus, H. strumo-
sus, H. maximiliani, H. nuttallii and H. grosseserratus have been produced
and used as a bridge to transfer resistance to broomrape race F (Jan and
Fernández-Martı́nez 2002). Positive results of transfer of rust resistance
genes from H. hirsutus and fertility restoration genes for a CMS cytoplasm
derived from H. giganteus have also been achieved using interspecific amphi-
ploids (Jan and Zhang 1995; Jan 2004).
6 Sunflower 181
Mutagenesis
Mutagenesis has been used successfully to generate genetic variability for useful
traits for the improvement of sunflower. This method is especially important if
variation for a given trait is not found in germplasm collections. Useful mutants
with short plant height, high oil and protein content and low hull content have
been obtained in sunflower using both irradiation and chemical treatments
(Voskoboinik and Soldatov 1974; Leclercq 1985; Fernández-Martı́nez and
Domı́nguez 1988). Other useful variability has been obtained for rust resistance
(Lofgren and Rama Raje Urs 1982) and herbicide resistance (Bervillé et al.
1992). Jan and Rutger (1988) used streptomycin and mitomycin C to produce
22 cytoplasmic and 7 nuclear male sterile mutants.
Mutagenesis results in sunflower have been particularly successful in mod-
ifying seed oil quality traits. One of the most valuable mutants produced is the
high oleic acid mutant (>80%), produced at the All-Union Research Institute
of Oil Crops of the former USSR, after treatment with dimethyl sulfate (Solda-
tov 1976). High levels of either palmitic or stearic acid (>25%) were achieved
using chemical and physical mutagens (Ivanov et al. 1988; Osorio et al. 1995).
Mutants with increased levels of gamma tocopherol (>95%) have been isolated
following chemical mutagenesis (Velasco et al. 2004b). The mutagenic treat-
ment is usually applied to the seeds, which after treatment are named M1 seeds.
Mutants can be detected in the M2 generation. For fatty acid and tocopherol
profile, which are mainly under embryogenic control, mutants are detected
analyzing M2 half-seeds.
6.7.1.2 Methods for Improving Source Populations

The success of isolating inbred lines with good combining ability or other
desired characters by the standard procedure of inbreeding and selection
depends on the frequency of superior S0 plants in the source populations.
Recurrent selection is an effective method for increasing this frequency. Sun-
flower breeders have used several intrapopulation and interpopulation recur-
rent selection methods to develop improved source populations. Most relevant
has been the use of the Pustovoit’s Method of Reserves described below, which
is a modified method of recurrent selection extensively used for developing
open-pollinated cultivars.
Two types of recurrent selection have been considered in sunflower, pheno-
typic recurrent selection, in which the phenotype of the S0 plant is the base of
selection, and genotypic recurrent selection, in which some type of progeny test
forms the base of selection. Phenotypic recurrent selection has been used by
sunflower breeders to improve populations for several traits, including yield, oil
percentage and disease resistance (Fick 1978; Miller 1987). For hybrid breeding,
female (B) and restorer (R) populations are generally managed separately to
simplify the development of inbred lines. The initial populations are often
a combination of high-performing inbred lines or a composite of lines from
one or several open-pollinated cultivars (Miller 1987). The parents selected to

form the initial population are crossed to produce a random-mated population.
Random-mating is accomplished by emasculating, either by hand or using
gibberellic acid, plants of each line and pollinating them with pollen gathered
randomly and equally from other lines. The source material or initial C0
material is grown and individual plants are selected and selfed. In the following
generation, the progenies of selected plants are sown in a separate row.
The progenies (rows) are crossed in all possible combinations and the resulting
hybrid seeds are bulked to form the C1 population. This completes the first cycle
of selection. The C1 plants from the first cycle are used for the second cycle of
selection. Phenotypic recurrent selection has been successfully used in sun-
flower for different traits, for example, self-incompatibility (Kinman 1970),
resistance to Sclerotinia stalk and head rot (Vear and Tourvieille 1984), seed
yield (Gundaev 1971), and oil content (Luduena et al. 1992).
Genotypic recurrent selection methods utilizing S1 progeny or testcross
evaluation have been effectively used in sunflower hybrid breeding to improve
yield and combining ability (Fick 1978) and drought resistance (Fernández-
Martı́nez et al. 1990). The S1 progeny recurrent selection method can also be
used to develop improved open-pollinated cultivars (Miller 1987). With this
method, several hundreds of individual S0 plants of the initial source C0 popu-
lation are selected and self-pollinated. In the second generation, part of the seed
is grown and evaluated for the traits of interest in replicated trials. Selected S1
progenies are recombined to form the C1 population, which is accomplished by
random mating plants obtained from reserve S1 seed. The recombination gen-
eration may be obtained in a greenhouse or winter nursery, which permits the
completion the three generations of a cycle of selection in 2 years. By using
a winter greenhouse nursery, spring evaluation, and recombination as a second
crop in the fall, Fernández-Martı́nez et al. (1990) were able to complete one
cycle per year in southern Spain.
In the test cross or half-sib progeny recurrent selection, test crosses instead of
S1 progenies are evaluated. Selected plants in the C0 initial source population
are selfed and simultaneously crossed with a tester the first year. These two
operations can be done in single-headed maintainer plants by self-pollinating
half of the head and emasculating and crossing the other half. In the case of
multi-headed restorer plants, the main head is emasculated and crossed,
whereas some secondary heads are selfed (Fick and Miller 1997). The type of
tester used depends on the objectives of selection. For example, in selection for
general combining ability (GCA) a broad base heterogeneous unrelated popu-
lation is used as tester. There are not many reports on half-sib recurrent
selection in sunflower breeding. Fick (1978) reported an increase of 3.5% in
seed yield after one cycle of recurrent selection in an R-line population using
testcross evaluation.
Interpopulation recurrent selection methods have also been used in sun-
flower. The reciprocal full-sib selection method, based in the production of
hybrid (full-sib) progenies and selfed seed in the same plant, was adapted for
6 Sunflower 183
sunflower using a multi-headed restorer (R) and a single-headed maintainer (B)

population (Miller and Fick 1978). The method is initiated by forming the
R and B populations by random mating diverse collections of R and B lines
to create the C0 populations. Full-sib progenies are obtained by transferring
pollen from a selected plant in the B line population to the emasculated main
head of a plant in the R line population. Self-pollinated seed is obtained by self-
pollinating secondary heads of the R line plant. The B line plant selected is also
self-pollinated. Full-sib hybrids are tested the following year in replicated yield
trials. On the basis of these evaluations, the self-pollinated S1 seed from the best
full-sib hybrids are bulked within each population and randomly mated in
a greenhouse or winter nursery to form the C1 populations to be used to start
a new cycle of selection. A cycle of selection is usually accomplished in 2 years
(Miller 1987), or even in 1 year (Fernández-Martı́nez et al. 1989a). Miller and
Hammond (1985) reported a 6.3% increase in yield per cycle using this scheme
of selection. Fernández-Martı́nez et al. (1989a) used the reciprocal full-sib
selection method to maximize seed yield under environments prone to drought,
with evaluations conducted under both irrigated and rainfed conditions.
6.7.1.3 Methods for Improving Open Pollinated Cultivars

Mass Selection
Mass selection implies the selection of individual plants from a population on
the basis of their phenotype for the improvement of a cultivar or population for
some specific traits. Seeds of the selected plants are mixed and planted the next
generation in order to obtain new cultivars or to maintain the varietal purity of
existing cultivars. Two methods of mass selection have been used in sunflower:
phenotypic mass selection and family selection. Phenotypic mass selection was
commonly used for improvement of sunflower during the early stages of culti-
var development in the former USSR. Several important cultivars were devel-
oped using this procedure (Gundaev 1971).
The family selection method is a form of mass selection that involves selec-
tion of individual S0 open-pollinated plants and classification of those plants for
characteristics of interest. Each plant is harvested and its seed is bulked with
other plants of phenotypically similar families. The various bulks are planted in
isolation for cross pollination. Gundaev (1971) listed many cultivars devel-
oped by this method. Mass selection has also been used in Argentina (Luciano
and Davreux 1967) and Mexico (Robles 1982) to produce improved sunflower
cultivars. Mass selection is a simple and economical method of selection but
its effectiveness depends on the heritability of the traits. In general, this
method has not been effective in sunflower breeding for traits with low
heritability such as seed yield, but it has been effective for improving other
traits including earliness, oil content or insect and disease resistance (Morozov
1947; Vrânceanu 1974).
Head to Row Selection (Pustovoit’s Method of Reserves)

The ‘‘method of reserves’’ was the most widely and successfully used method
to develop open pollinated sunflower cultivars. It was developed in the former
USSR by V.S. Pustovoit during the 1920s (Pustovoit 1967). The method is
a form of recurrent half-sib selection that includes progeny testing and sub-
sequent cross pollination among superior progenies (Alexander 1963).
The ‘‘method of reserves’’, as outlined by Pustovoit (1967) is initiated by
forming a heterogeneous population of germplasm including elite cultivars,
intercross hybrids, and world collection entries. About 10,000–15,000 plants
are selected from this population with the main criterion that plants have
500–2,000 seeds per head. The seeds are analyzed for hull and oil percentage
and around 1,200–1,500 heads are selected for progeny evaluation. Half-sib
seeds of these S0 selections are evaluated for agronomic, disease resistance,
and seed quality traits in single row plots with two replications. A check
consisting of the best existing cultivars is included in every third plot as
a control. On the basis of the observations from the first year of testing,
15–20% of the S0 selections are planted in a second-year observation nursery.
Remnant half-sib seed produced in season 1 is utilized also to plant this
nursery and to test for disease resistance.
Based on performance in the first and second year nurseries, the original seed
of the best 20–50 S0 plants selected in season 1 are planted in a cross pollination
nursery for random mating using an isolation distance of 200–300 m between
nurseries. Undesirable phenotypes including disease infected, extremely tall or
branched plants are removed. Seeds from random-mated plants are analyzed
for oil content and the seeds of selected plants are bulked for a new cycle of
selection or for testing at a larger scale. After three years of testing, superior
populations are released as new open-pollinated cultivars. The ‘‘method of
reserves’’ has been especially useful in increasing oil content and oil yield per
hectare, developing early maturity, and resistance to diseases, Orobanche, and
sunflower moth (Gundaev 1971). In the former USSR, cultivars developed by
this method were grown in 4.6 million hectares in 1973 (Pustovoit and Gubin
1974), and it was successfully used in other countries such as Romania (Vrân-
ceanu 1974) and former Yugoslavia (Škorić 1988). However, in spite of its
success, the genetic gain per year in comparison with other recurrent selection
methods is limited due to the number of years used for evaluation at each cycle
of selection.
6.7.1.4 Methods for Improving Hybrid Cultivars

Inbreeding for improving sunflower was used by early Russian and Canadian
breeders to isolate uniform strains with variation for different traits such as
plant height, oil content, disease, and pest resistance (Voskoboinik and Solda-
tov 1974; Unrau 1947). However, the main value of inbreeding was to develop
true breeding lines with desirable characteristics to be used in the production of
6 Sunflower 185
synthetic cultivars or hybrids. The first results involving hybridization of inbred

lines showed significant heterosis for seed yield and other traits (Unrau 1947;
Putt 1962).
The most common method for developing new inbred lines in sunflower is
pedigree selection, but bulk selection and single seed descent have also been
used. Backcrossing is frequently used for modifying existing lines. The germ-
plasm sources are derived from open-pollinated source materials, populations
improved by recurrent selection, or from planned crosses between inbred lines
(Miller 1987).
Pedigree selection involves self-pollination of phenotypically desirable plants
in the F2 or S0 generation, depending on the starting material. Selection is
practiced for agronomic type, disease resistance, or other desired traits. The
F3 progeny of each F2 plant is grown the next season. Plants are selected and
self-pollinated within the best F2:3 lines. The process of inbreeding and selection
is continued for five generations. In the F3 generation, pollen from selected
plants within the F3:4 lines may be used to cross to tester lines to produce
testcross seed. The hybrid testcrosses are evaluated and F4:5 lines with superior
combining ability can be selected. In the case of B lines, pollen can be collected
to cross to a CMS source to begin the conversion of the F4-derived B line to
CMS (A line) by the backcross method. This process generally requires five
backcrosses using the F4-derived line as recurrent parent.
The development of lines by the backcross method is probably used more
frequently than any other method, except pedigree selection. Backcrossing is
used in the context of transferring a trait from one genotype (donor parent) to a
desirable genotype (recurrent parent). The trait being transferred is usually
simply inherited. Backcrossing is usually a correctional breeding method that
is used to enhance the performance of an elite inbred line, but it is also used to
introgress a specific gene in an elite inbred line. If the backcrossed-derived line
must be essentially identical to the recurrent parent, about six backcrosses must
be made. The complexity of the genetic transference of a given trait to a
recurrent parent depends on the number of genes involved, their dominant or
recessive nature, and the presence or absence of maternal effects. Examples for
dominant traits controlled by one gene involve the transfer of the high oleic trait
to the sunflower lines HA 89 and RHA 271 (Fernández-Martı́nez et al. 1993)
and the resistance of downy mildew to HA 89 (Miller and Gulya 1988). These
traits can be easily transferred because plants or seeds with the desired trait can
be identified in the F1 backcross generation. However, in many cases the target
traits are recessive and do not show up in the F1 generation, which increases the
duration of the backcross generation. Such a limitation can be overcome by
using marker-assisted selection, which allows the recessive genes to be identified
in the F1 backcross generation.
The final value of an inbred line is determined by testing general or specific
combining ability in hybrid combinations. For general combining ability, the
choice of the tester (homogeneous inbred lines vs. heterogeneous sources)
depends on the specific program objectives. The most common testers are
inbred lines that are being used in commercial hybrids. Miller et al. (1980) and
Domı́nguez and Fernández-Martı́nez (1987) found that inbred lines are effec-
tive in identifying lines with the best combining ability. Evaluation for combin-
ing ability often begins at the S3 or S4 generation, but a system of early
generation testing beginning after the first generation of selfing was reported
to be effective in identifying lines with good combining ability (Shein 1978).
Inbred lines are used primarily in the production of single crosses or three-
way sunflower hybrids using the cytoplasmic male sterility and the fertility
restoration system. Single-cross hybrids are produced by crossing a male-sterile
female (A line) with a male-fertile restorer (R line). A three-way hybrid is made
by crossing an A line with an unrelated maintainer line (B line) to produce
a male-sterile single-cross hybrid. This hybrid is crossed with an R line to
produce a male-fertile, three-way hybrid. Generally, single crosses are higher
yielding than three-way hybrids and have greater uniformity (Fick and Zimmer
1976; Miller 1987). Three-way hybrids are produced primarily to reduce seed
cost, since seed yield of single-cross female parents is often 1.5–2.0 times greater
than that of inbred lines, although inbred lines that yield up to 80% of their
hybrids have been developed (Fick 1978; Škorić 1988). Three-way hybrids are
considered more stable over environments than single crosses due to their
greater heterogeneity (Fick and Zimmer 1976; Schuster and Friedt 1988). The
use of double-cross hybrids in sunflower to further improve adaptation and
yield stability has also been suggested (Vulpe 1974).
Inbred lines have also been used to produce synthetic varieties in Canada
(Putt 1966) and in the former USSR (Voskoboinik and Soldatov 1974). Putt
(1966) concluded that high-yielding synthetics could be developed from as few
as three to five inbred lines. Synthetic cultivars have been evaluated with
favourable results in countries where hybrid production is not practical, for
example Nigeria (Ado et al. 1991) and Egypt (Shabana 1990).
6.7.1.5 Methods for Producing Hybrid Seed

Efficient and economical production of hybrid seed at a commercial scale was
greatly facilitated by the discoveries of genetic and cytoplasmic male sterility
(Leclercq 1966, 1969). Nuclear male sterility was used to produce commercial
hybrid seed in the early 1970s taking advantage of a close linkage between genes
for male sterility and anthocyanin pigmentation of the plants, which allows the
identification and removal of male-fertile plants (Leclercq 1966; Vrânceanu
1974). Detailed methods of hybrid seed production using this system have
been described by Vrânceanu (1974) and Škorić (1988). However, the use of
nuclear male sterility to produce hybrid seed has been replaced by the cytoplas-
mic male sterility and fertility restoration system and, although new sources of
cytoplasmic male sterility have been found, the source discovered by Leclercq
(1969) is used almost exclusively in current hybrid seed production programs.
This system requires a cytoplasmic male-sterile line (A line), which is main-
tained by crossing to a genetically identical male-fertile line with a fertile
6 Sunflower 187
cytoplasm (B line), and a restorer line (R line) which combines well with the A
line and restores the fertility in the hybrid cultivar. The seed harvested from the
A line is grown commercially as a hybrid cultivar.
6.7.2.1 Procedures for Selfing and Artificial Hybridization

In order to carry out selfing and controlled crosses, sunflower heads must be
isolated from insect pollination. Paper and cloth bags are most commonly used.
Paper bags cost less and may be satisfactory in some environments if excessive
rain does not occur during the latter part of the growing season, but cloth bags
are more desirable for the standpoint of seed set and durability (Vrânceanu
1974; Roath and Pomeroy 1988). Artificial hybrids are produced by emascula-
tion of the female parent followed by pollination with pollen of the desired male
parent. Hand emasculation is commonly used, but chemical emasculation has
also been extensively used. Hand emasculation involves removing the anthers of
the disk flowers with forceps early in the morning before the anthers have
dehisced and before the stigmas have elongated up through the anther tubes.
A large head will flower over a five to six-day period with three to six rings of
florets flowering each day. Stigmas will be fully elongated and receptive 1–2 h
after emasculation and will remain receptive 4–5 days. Chemical emasculation
with gibberellic acid (GA3) is utilized by sunflower breeders to produce hybrid
seed. A 50 to 100-ppm concentration of GA3 is applied to sunflower buds of
approximately 1–1.5 cm diameter with generally good results (Miller and Fick
1978). However, cultivars and inbred lines may show different responses to GA3
requiring higher or lower concentrations and earlier or later application times
(Piquemal 1970). Various negative effects such as incomplete male sterility,
reduced female sterility, and stem elongation have been associated with the
use of GA3, depending on concentration and timing of application (Miller
1987).
Pollination can be accomplished by several methods. If the male plant is
located adjacent to the female parent and is shedding pollen, the receptacles can
be rubbed together to transfer the pollen from the anthers of the male to the
stigmas of the female plant. However, the most common technique is to collect
pollen from the male parent in paper bags or on a cloth or leaf from heads
isolated with bags prior to flowering. Pollen in paper bags can be stored up to 4
weeks in a refrigerator at a temperature of 4–68C and relative humidity of less
than 4%, and several years at –768C (Frank et al. 1982).
6.7.2.2 Techniques Used for Interspecific Hybridization

Direct crosses of annual wild species, except H. agrestis with cultivated sun-
flower are possible using conventional methods. However, crossing perennial
species, including diploids, tetraploids and hexaploids, is much more difficult

and also produces sterile F1 plants, requiring special techniques (Jan 1997).
The two-step embryo culture procedure developed by Chandler and Beard
(1983) avoids embryo abortion and seed dormancy and facilitates interspecific
hybridization. With this technique, embryos are excised and cultured 3–7 days
after pollination. The embryos are cultured in Petri dishes on an appropriate
solid growth medium (Chandler and Beard 1983). For embryo germination
and seedling growth, the cultured enlarged embryos are transferred after 1–2
weeks to a liquid germination medium in test tubes. An optimized method for
culturing difficult hybrid embryos derived from perennial Helianthus species
was proposed by Jan (1988), and additional modifications have been sug-
gested (Kräuter et al. 1991).
Another technique to facilitate interspecific transfer is the use of induced
polyploidy by chromosome doubling using colchicine applied to apical meris-
tems. Jan (1988) described a modified colchicine chromosome doubling techni-
que with a significant positive effect on backcross seed set. The apical meristems
are submerged in a 1.5 g/kg colchicine solution with 2.0 g/kg DMSO (dimethyl
sulfoxide) for 5 h in the dark. Then, chromosome doubling of each head is
verified by pollen grain size and stainability.
6.7.2.3 Field Plot Techniques for Cultivar Evaluation

Newly developed hybrids or open pollinated populations must be evaluated
before they can be recommended for releasing. Detailed procedures of field
testing sunflower genotypes have been described by Miller (1987), Fick and
Miller (1997), and Vrânceanu (1974). The testing process largely depends on the
genetic control of the traits. For qualitative traits controlled by major genes and
scarcely influenced by the environment, limited evaluations in the greenhouse
or field may be adequate. For quantitative traits, such as yield or oil percentage,
lines or cultivars must be tested over years and locations. A common plot size
includes two to five rows from 6 to 10 m length spaced 0.75 m. Usually test plots
are overplanted and thinned to a uniform stand soon after emergence.
Preliminary yield evaluations are usually planted in plots with one or two
unbordered rows with two to three replications at one or more locations using
a simple lattice or augmented design. Check cultivars are sown every five to ten
rows in the augmented design and randomly in the lattice design. Advanced
yield trials are planted in plots with two to five rows with three to four replica-
tions at several locations. In the case of three to five rows per plot, only the
center rows are harvested. Significant interplot competition effects may occur
between cultivars showing wide differences in height and maturity (Domı́nguez
et al. 1980). In these cases, the effect of interplot competition can be reduced by
grouping cultivars by height and maturity. The most common experimental
designs for advanced yield trials are randomized complete block design and
simple lattice, the latter used when the number of entries exceeds 36.
6 Sunflower 189
6.7.2.4 Techniques Used for Greenhouses and Off-Season Nurseries

Greenhouse and off-season nurseries are frequently used by sunflower breeders
to grow several generations per year and speed up the breeding program.
Postharvest dormancy is a frequent problem in greenhouse and off-season
plantings. Fick (1978) reported a technique to overcome dormancy consisting
in soaking the seeds in a concentration of 0.6 mL ethrel/L of water for 16 h.
Treatments with grow retardants to reduce the height of plants grown in the
greenhouse and produce plants with short, thick stems that are easy to manage
have been used for winter planting in the greenhouse (Fick and Miller 1997).
Another technique that may have value in programs requiring short generation
time is the application of growth regulators as desiccants (Rana et al. 1990).
6.7.2.5 Laboratory Techniques for Seed Quality Evaluation

Breeding programmes to improve seed oil quality traits require the availability of
adequate screening techniques to measure them. In sunflower, both the fatty acid
and the tocopherol profile of the seeds are mainly under gametophytic control,
i.e. they are governed by the genotype of the developing embryo. Accordingly,
selection for these oil quality traits can be conducted at the single-seed level.
Nondestructive methods to measure these traits in single seeds have been devel-
oped. Downey and Harvey (1963) developed the half-seed technique for non-
destructive analysis of the fatty acid composition of single seeds of rapeseed
(Brassica napus L.). The technique has been adapted to sunflower (Conte et al.
1989). It consists of the removal of a small seed portion in the region extremely
distal to the embryo in a way that the germination capacity of the seed is not
affected. The excised half seed is used for chemical analysis whereas the other half
seed containing the embryo can be sown to produce a viable plant. The half-seed
technique has been used for the nondestructive analysis of fatty acid composition
(Conte et al. 1989), tocopherol composition (Demurin et al. 1996) and total
tocopherol content (Velasco et al. 2004b).
The use of near infrared reflectance spectroscopy (NIRS) has facilitated the
screening for seed quality traits in sunflower. NIRS is a fast, nondestructive and
cost-effective technique that permits the simultaneous analysis of multiple con-
stituents in a single measurement. This requires the previous development of
individual calibration equations for every constituent to transform NIRS spectral
data into chemical information. NIRS was first applied to determine oil content
in sunflower meal samples (Robertson and Windham 1981). The technique has
been also used to analyse protein content, fiber content (Kaffka et al. 1982),
tocopherol content, phytosterol content (Gotor et al. 2007), and free fatty acid
content (Moschner and Biskupek-Korell 2006) in sunflower meal. However, the
application of NIRS to sunflower breeding requires the use of small samples of
intact achenes. Sato et al. (1995) used NIRS for the analysis of oil content in
hulled sunflower seeds. Pérez-Vich et al. (1998) combined the simultaneous
analysis of seed oil content and fatty acid profile in both intact and hulled
sunflower kernels.
Selection for seed quality at a single seed level has been facilitated by the use
of near-infrared spectroscopy (NIRS) for analyzing the fatty acid profile of
intact or hulled individual kernels. Sato et al. (1995) demonstrated the feasi-
bility of NIRS for measuring the concentration of linoleic acid in the oil of
single hulled kernels of sunflower. Velasco et al. (1999) reported that NIRS
permitted the discrimination of intact achenes for oleic and linoleic acid con-
centration in the seed oil. Velasco et al. (2004c) used NIRS for large-scale
screening for high stearic acid concentration in hulled sunflower seeds.
6.7.2.6 Techniques for Disease Resistance and Broomrape Evaluation

Breeding for disease resistance requires the creation of a disease environment to
differentiate between resistant and susceptible plants. Breeders and plant
pathologists have developed effective procedures to evaluate breeding materials
for most of the major sunflower diseases. These techniques have been described
in detail by Škorić (1988), Gulya et al. (1997) and Fick and Miller (1997).
Downy mildew evaluations are sometimes conducted in lands naturally
infected with the pathogen, but more often under controlled conditions in
greenhouse or growth chamber. The procedure described by Zimmer (1974) is
highly effective and is extensively used in breeding programs. It consists of the
inoculation of germinated hulled seeds, with radicals 10–20 mm long, with a
suspension of 10,000 or more zoosporangia per ml of distilled water for 18 h
at 208C. Then, the inoculated seeds are planted in sterile soil and maintained
on greenhouse benches during 14 days at 20–258C and 16 h day length
photoperiod. Susceptibility is indicated by the occurrence of sporulation of
the fungus on the cotyledons or the under-surface of the first true leaves after
18 h in a saturated humidity chamber. A PCR test has been developed to
detect the presence of the pathogen in sunflower seeds (Ioos et al. 2007).
Researchers working with sunflower rust adopted much of the methodology
developed by cereal rust pathologists. Techniques for spore collection and
storage and inoculation under greenhouse and field conditions were reviewed
by Gulya and Marisevic (1996). Greenhouse evaluations for sunflower rust are
conducted on seedlings after inoculating with 10:1 mixture of talc and uredios-
pores. Field evaluations are conducted by spraying 3 to 4-week-old susceptible
border row plants with water suspension of urediospores collected from com-
mercial fields the preceding season. Plants are sprayed in the evening when
temperatures are lower and then covered overnight with metal or plastic con-
tainers to provide a high level of humidity. Infection of susceptible plants
provides natural inoculum and subsequent spread of rust throughout the
nursery allowing the selection of resistant plants (Fick 1978).
The most common procedure of evaluation for resistance to Sclerotinia stalk
and head rot is field testing in naturally infected fields (Gulya et al. 1997), but
the results depend on soil and environmental factors. Several field and green-
house methods have been developed. They involve adding sclerotia or mycelia-
infested cereal grains to the soil at planting time or directly to the basal stem to
6 Sunflower 191
increase the level of infection (Pirvu et al. 1985; Rashid 1992) or immersing the
roots of 3-week-old plants in a Sclerotinia culture filtrate (Huang and Dorrell
1978). For head rot resistance, an effective procedure involving spraying an
ascospore suspension onto the florets and covering the head with a paper bag
was developed (Tourvieille et al. 1978).
Evaluations for Phomopsis stem canker caused by Diaporthe helianthi are
best conducted in fields under intensive natural infection (Fick and Miller
1997). In the absence of high levels of natural infection, several artificial
inoculation methods have been described consisting of placing mycelial
explants on mature leaves, stem or petioles (Tourvieille et al. 1988), spraying
ascospores on the leaves (Marisevic and Gulya 1992), or artificially infecting
field plots by placing contaminated stalk segments in the field followed by
sprinkler irrigation (Griveau et al. 1992).
Effective evaluation methods have also been developed for screening for
resistance to other sunflower diseases such as Verticillium wilt, Alternaria,
Phoma black stem and Rhizopus root rot. Field evaluation methods in naturally
infested plots and artificial inoculation procedures have been reviewed in detail
by Škorić (1988) and Gulya et al. (1997). Rani and Ravikumar (2007) suggested
a combination of gametophytic and conventional sporophytic selection to
improve selection efficiency for partial resistance to Alternaria leaf blight.
Evaluations for Orobanche resistance can be conducted in naturally infested
fields, but more frequently breeders use artificial inoculation with seeds of the
parasite collected in previous years (Škorić 1988). These evaluations are con-
ducted in artificially infected fields or in pots in greenhouses and growth
chambers. A soil mixture (sand: silt, 1:1) is homogeneously infested with
broomrape seeds adding 250 mg of seeds per kg of soil (Panchenco 1975).
Sunflower seedlings are planted in peat pots with the inoculated soil mixture
and incubated in a growth chamber under controlled conditions of light and
temperature during 15–20 days and then transplanted to pots in the greenhouse
or to the field (Škorić 1988). Resistance or susceptibility is determined by the
percentage of sunflower plants that are parasitized and the average number of
broomrape plants per sunflower plant. Special procedures have been developed
for non-destructive in situ monitoring the developmental stages of the parasite
and its interaction with sunflower (Eizenberg et al. 2005).
Despite the tremendous economic significance of sunflower, initial molecular

research on this crop was considerably delayed in comparison to other crops of
similar or even lower importance. In fact, the first reports on molecular markers
development in sunflower emerged nearly a decade after the initial studies of
restriction fragment length polymorphism (RFLP) mapping in plants.
However, molecular research in sunflower has been considerably stimulated
in recent years by significant contributions in the construction of saturated
genetic linkage maps, mapping and characterization of genes controlling

important traits, and understanding its genetic make up. Conversely, transgenic
approaches have been scarcely afforded. There is still a huge amount of inno-
vative research to be conducted in sunflower, but many tools are being con-
tinuously developed and are available to sunflower breeders.
6.8.1 Genetic Markers and Genetic Linkage Maps in Sunflower
In sunflower, as in other plant species, genetic markers were originally used

in genetic mapping to determine the order of the genes along chromosomes, and
evolved from morphological markers through isozyme markers to DNA mar-
kers. The latter have evolved from hybridization-based detection to polymerase
chain reaction (PCR) amplification and, most recently to sequence-based sys-
tems. Both morphological and isozyme markers are limited in number.
Additionally, the morphological markers are affected by the environment,
and a given marker can affect other morphological traits because of pleiotropic
gene action. Consequently, genome-wide analysis is not feasible using only
morphological and isozyme markers. DNA markers are typically derived from
a small region of DNA that shows sequence polymorphism between individuals
within a species, and may be classified into random DNA markers (also known as
anonymous or neutral markers), gene-targeted markers (also known as candidate
gene markers) and functional markers. Random DNA markers are derived at
random from polymorphic sites along the genome, whereas gene-targeted mar-
kers are derived from polymorphisms within genes. Finally, functional markers
are derived from polymorphic sites within genes causally associated with pheno-
typic trait variation. In this section, we will give an overview of DNA marker
development and mapping in sunflower. Functional genetic linkage maps created
for mapping phenotypic and quantitative trait loci will be preferentially described
in a later section. Linkage group nomenclature that will be used is that of the
reference public map of Tang et al. (2002).
6.8.1.1 Random DNA Markers and Maps Based on them

Restriction Fragment Length Polymorphism (RFLP) Markers
In sunflower, the first DNA-based markers developed were RFLP markers.
Most of the published sunflower RFLP markers were developed with anon-
ymous cDNA clones, which yield low copy, polymorphic restriction fragments
(Berry et al. 1994; Gentzbittel et al. 1994; Jan et al. 1993). RFLP markers were
initially mapped in cultivated sunflower by different research groups, and
genetic maps based on them were reported. The maps by Berry et al. (1995)
and Jan et al. (1998) were based on individual F2 populations, whereas those of
Gentzbittel et al. (1995, 1999) and Berry et al. (1996) were composite maps
based on data of different mapping populations (Table 6.6). In general, these
Table 6.6 Characteristics of sunflower linkage genetic maps ordered according to marker system and date of publication
Average
No. of No. of Map interval
6 Sunflower
Population No. of mapped Linkage length size

Reference Mapping populations type individuals Markers loci groups (cM) (cM)
Berry et al. HA89 x ZENB8 One F2 289 RFLP 234 17 1380 5.9
(1995)
Gentzbittel et al. HA89 x RHA266; CX x Three F2 560 RFLP 237 23 1150 7
(1995) RHA266; PAC2 x and two
RHA266; (HA89 x BC1F1
CX) x HA89; (HA89 x
CX) x CX
Berry et al. ZENB8 x HA89; Nine F2 1287 RFLP 635 17 1472 2.3
(1996) ZENB8 x PAC2;
ZENB8 x ZENR7;
BSA52 x RHA297;
HA89 x RHA271 ;
ZENR7 x RHA801;
HA89 x ZENR9;
ZENR1 x ZENR8;
ZENB4 x HA300
Jan et al. (1998) RHA271xHA234 One F2 93 RFLP 271 20 1164 4.6
Gentzbittel et al. HA89 x RHA266; CX x Seven F2 1115 RFLP 238 17 1534 6.7
(1999) RHA266; PAC2 x
RHA266; SD x PAC1
; SD x CP73; CP73 x
PAC1 ; GH x PAC2
Ungerer et al. (H. anomalus ANO- 56 RAPD, AFLP, isozyme 701 17 1983 6.2
(1998) 1497 x H. anomalus
ANO-1506) x CMS89
193
194
Average
No. of No. of Map interval
Population No. of mapped Linkage length size
Reference Mapping populations type individuals Markers loci groups (cM) (cM)
Peerbolte and CX x RHA266; PAC2 x Two F2 184 RFLP, AFLP 437 19a 1144
Peleman RHA266 from
(1996) Gentzbittel et al.
(1995)
Gedil et al. (1999) HA370 x HA372 One F2 108 RFLP, AFLP 400 17 1326 3.3
Flores-Berrios PAC-2 x RHA-266 One RIL 99 AFLP 264 18 2558 9.9
et al. (2000)
Langar et al. HA89 x LR4 One R9 171 DALP, AFLP 305 18 2169 6.1
(2003) RIL
Tang et al. (2002) RHA280 x RHA801 One R7 94 SSR 459 17 1368 3.1
RIL
Yu et al. (2003) HA370 x HA372 from One F2 94 RFLP, SSR 202 17 1275 6.3
Gedil et al. (2001a)
Yu et al. (2003) RHA280 x RHA801 One R7 94 SSR, 577 17 1423 2.5
from Tang et al. RIL INDEL
(2002)
Yu et al. (2003) PHA x PHB One RIL 94 SSR 264 20 1199 4.5
Rachid Al- PAC-2 x RHA-266 from One RIL 123 AFLP, SSR 367 21 2916 7.9
Chaarani et al. Flores-Berrios et al.
(2002) (2000)
Lai et al. (2005) RHA280 x RHA801 One RIL 94 SNP, SSR 439 17 1349
from Tang et al.
(2002)
Hu et al. (2004) 83HR4 x RHA345 One RIL 129 TRAP 160 17b 1140 9.0
Hu (2006) RHA280 x RHA801 One F7 RIL 92 TRAP, SSR 760 17 1747
from Tang et al.
J.M. Fernández-Martı́nez et al.
(2002)
a
The authors describe 19 stable linkage groups (3 markers or more) in a total number of 23 linkage groups.
b
The authors describe 17 linkage groups and 4 pairs of markers not assigned.
6 Sunflower 195
maps comprised 17 or more linkage groups that presumably correspond to the

17 haploid chromosomes of sunflower, and covered distances close to the
estimated length of the sunflower genome (1,650 cM; Gentzbittel et al. 1995)
(Table 6.6). Distorted segregation and duplicated RFLP loci were detected by
Berry et al. (1995, 1996) and Gentzbittel et al. (1995).
Random Amplified Polymorphic DNA (RAPD) Markers

Despite the dominant and low reproducible nature of random amplified poly-
morphic DNA (RAPD) markers, they were used in early genetic studies in
sunflower. High levels of RAPD variation were reported in sunflower species
(Lawson et al. 1994; Teulat et al. 1994) with the proportion of polymorphic loci
averaging more than 50% for most domesticated lines. Methods such as bulked
segregant analysis (BSA) allowed the rapid identification of RAPD markers
associated with agronomically important traits in sunflower, such as rust
resistance (Lawson et al. 1996) or broomrape resistance (Lu et al. 2000). To
overcome RAPD limitations, RAPD bands can be converted into sequence-
characterized amplified region (SCAR) markers. In sunflower, Lawson et al.
(1998) developed SCAR markers from RAPD bands linked to two rust resis-
tance genes, demonstrating their robustness for the detection of these resistance
genes in different genetic backgrounds, and Lu et al. (2000) reported SCAR
markers linked to the Or5 gene conferring resistance to race E of broomrape.
RAPDs have been used for mapping in sunflower, particularly in wild
species. Rieseberg et al. (1993) constructed a Helianthus anomalus map based
on 161 RAPD markers and one isozyme locus. Later on, this map was expanded
and now includes 549 RAPD, 151 AFLP, and one isozyme locus (Rieseberg
et al. 1995; Ungerer et al. 1998), covering 17 linkage groups and 1,983 cM
(Table 6.6). RAPD maps were also developed for wild H. annuus and
H. petiolaris (Rieseberg et al. 1995), based on 212 and 400 RAPD loci, respec-
tively. These authors reported 17 linkage groups for both species covering
1,084 cM for H. annuus and 1,761 cM for H. petiolaris.
Amplified Fragment Length Polymorphism (AFLP) Markers

AFLP are powerful markers for genome mapping and genetic variability stu-
dies, since they are highly reproducible, require no prior sequence information,
and have a high multiplex ratio. However, AFLP markers are typically domi-
nant and therefore their utility is greatest for projects where dominance is not
disadvantageous. AFLP markers have been used to fingerprint elite sunflower
inbred lines (Hongtrakul et al. 1997), to construct new genetic maps, and to
increase the density and to fill gaps of already developed genetic maps. Peer-
bolte and Peleman (1996) added 291 AFLP loci to two of the F2 populations
used by Gentzbittel et al. (1995) (Table 6.6). These markers pulled two linkage
groups together and permitted several previously unlinked RFLP marker loci
to be mapped (Knapp et al. 2001). Gedil et al. (2001a) added 296 AFLP loci to a
104 RFLP loci map based on markers from Berry et al. (1996) and Jan et al.
(1998), and constructed an AFLP-RFLP map that comprised 17 linkage
groups, had a mean density of 3.3 cM, and was 1,326 cM long. Other AFLP
maps have been developed. Flores-Berrios et al. (2000) constructed an AFLP
map of 2,833.7 cM using 99 recombinant inbred lines (RILs) (Table 6.6), which
was later improved with additional AFLP markers (Rachid Al-Chaarani et al.
2002). Langar et al. (2003) constructed a genetic map 2,169 cM long combining
direct amplification of length polymorphism (DALP) markers and AFLP
markers (Table 6.6).
Simple Sequence Repeats (SSRs) or Microsatellites

Microsatellite markers are ideal DNA markers for genetic mapping and popu-
lation studies because of their abundance, high levels of polymorphism, multi-
allelic nature, codominant inheritance, wide dispersion in genomes, ease of
assay using PCR, and ease dissemination across laboratories (Powell et al.
1996). Early studies demonstrated the presence of SSRs in the sunflower gen-
ome with (A)n, (GA)n, and (CA)n being the most abundant motifs (Dehmer
and Friedt 1998a). Later on, different research groups have described the
development and characterization of SSR markers (Gedil 1999; Paniego et al.
2002; Yu et al. 2002; Tang et al. 2002), summing up a total of 2,040 markers
(Paniego et al. 2007).
Tang et al. (2002) constructed the public-reference SSR map of sunflower
using 94 F7 recombinant inbred lines (RILs) and 408 polymorphic SSR markers
(Table 6.6). The map was 1,368 long and had a mean density of 3.1 cM. Yu et al.
(2003) provided the first sunflower cross-referenced maps by mapping 701 new
SSR and 89 RFLP or INDEL marker loci into three populations, two of them
previously used by Gedil et al. (2001a) and Tang et al. (2002) (Table 6.6). From
these maps, Tang et al. (2003a) developed a composite SSR linkage map of
sunflower that integrated 657 loci in a 1,423 cM map with a mean density of
2.2 cM per locus. This map allowed the selection of 95 single-locus SSRs at an
average spacing of 12.9 cM representing a near-genomewide collection for a
first-pass scan of the sunflower genome, from which 13 six-locus PCR multiplex
sets including 78 SSRs were developed. A different set of 78 SSR markers was
selected by Zhang et al. (2005) for sunflower variety identification and diversity
assessment.
The AFLP map developed by Rachid Al-Chaarani et al. (2002) was
improved by increasing the number of AFLP markers and integrating 38
SSR markers (Rachid Al-Chaarani et al. 2004). In the new map, 367 AFLP
and SSR marker loci were placed in 21 linkage groups covering 2,916 cM
(Table 6.6). An additional improvement of this SSR-AFLP map has recently
been reported by Paniego et al. (2007), who integrated 161 new SSR markers
from different sources, and cross referenced this map to the public SSR map
of Tang et al. (2002).
6 Sunflower 197
6.8.1.2 Gene-Targeted Markers and Maps Based on Them

Markers Based on Sequenced RFLP-cDNA Probes
INDEL (Insertion-Deletion) markers were developed from 81 RFLP markers
by sequencing the cDNA clones, aligning sunflower cDNA and Arabidopsis
genomic DNA sequences, predicting from such an alignment intron sites in
sunflower genes, and designing flanking primers to amplify the introns and
flanking coding regions spanned by the primer pairs (Yu et al. 2003). The
genetic linkage map position of these markers (ZVG1 through ZVG81) inte-
grated in the public SSR map of Tang et al. (2002) is described in Yu et al.
(2003). Recently, Kolkman et al. (2007) resequenced in different inbred lines
and wild sunflower populations essentially these 81 genes previously mapped as
RFLP markers and identified 1078 single nucleotide polymorphisms (SNPs)
and 178 INDELs.
Markers Based on ESTs

Expressed sequence tags (ESTs) are typically unedited, automatically pro-
cessed, single-read sequences produced from cDNAs, and are currently the
most widely sequenced nucleotide element from the plant genomes. Different
EST sequencing programs have been carried out in sunflower, including the
Compositae Genome Project, and other programs reported by Fernández et al.
(2003), Tamborindeguy et al. (2004), and Ben et al. (2005). These programs
have produced 94,111 EST entries for Helianthus annuus in GenBank (last
accessed October, 2007). A comprehensive annotated sunflower EST database
can be found at the database of the Compositae Genome Project (http://
compgenomics.ucdavis.edu/).
EST constitutes a novel source of DNA-based markers that are physically
associated with coding regions of the genome. In sunflower, EST resources
have been used to develop SNP and SSR markers. Pashley et al. (2006)
developed a novel suite of 48 polymorphic SSR markers surveying sunflower
EST sequences to identify those containing SSRs. These authors found that
SSRs based on ESTs exhibited higher transferability across species as com-
pared to anonymous SSRs. SNP/INDEL markers from sunflower ESTs
were developed by Lai et al. (2005). These authors identified 605 ESTs
that displayed SNP or small insertion-deletion variation in silico, had appar-
ent tissue-specific expression patterns, and/or were ESTs with candidate
gene function for development, cell transport, metabolism, plant defence,
and tolerance to abiotic stress. Primer pairs for 535 of these loci were
designed from the ESTs. Two hundred and forty-three of these markers
were mapped within a 196 SSR loci framework map from the RIL popula-
tion reported by Tang et al. (2002) and Yu et al. (2003). The SNP/INDEL-
SSR map was 1,349 long (Table 6.6), and constitutes the first functional map
based on sunflower ESTs.
Target region amplification polymorphisms (TRAPs) are derived from a

rapid and efficient PCR-based technique, which uses bioinformatics tools and
EST database information to generate polymorphic markers around targeted
candidate gene sequences (Hu and Vick 2003). The TRAP technique has been
employed in sunflower to construct a linkage map based on 160 TRAP markers
(Hu et al. 2004) (Table 6.6), and to define the sunflower linkage group ends
through the use of TRAP markers based on Arabidopsis-type telomere repeat
sequences (Hu 2006).
Sunflower ESTs have also been used as a source for developing universal
markers useful for comparative mapping and phylogenetic analysis within the
sunflower family, Asteraceae. Chapman et al. (2007) used alignments of a
conserved orthologous set of ESTs from sunflower and lettuce and genomic
sequences of Arabidopsis to design a suite of primer pairs that were conserved
across species, but which were predicted to flank introns (variable regions) and
therefore to detect polymorphism within species. One hundred and ninety-two
of these primer pairs were tested for amplification across eight diverse members
of the Asteraceae. From these, 85% amplified in at least one taxon, and 20%
amplified in all the eight taxa tested, the majority of these loci being poly-
morphic within species.
6.8.1.3 Functional Markers

Functional markers are derived from polymorphic sites within the genes
known to be causally involved in phenotypic trait variation. The devel-
opment of functional markers requires allele-specific sequences of func-
tionally characterized genes from which polymorphic, functional motifs
affecting plant phenotype can be identified. Functional markers have
been developed in sunflower for traits determining oil quality (Tang
et al. 2006b), or herbicide resistance (Kolkman et al. 2004). A detailed
description of these markers will be given in the section on molecular
breeding.
6.8.2.1 Germplasm Characterization

The characterization of genetic structures in cultivated sunflower was one of
the first aims of genetic fingerprinting using molecular markers. Initial studies
using RFLPs consistently separated sunflower inbred lines into sterility main-
tainer (B-line) and fertility restorer (R-line) groups (Gentzbittel et al. 1994;
Zhang et al. 1995), reflecting breeding strategies that maximize heterosis.
AFLP and SSR analyses confirmed these results (Hongtrakul et al. 1997;
Paniego et al. 2002; Yu et al. 2002). Tang and Knapp (2003) performed
6 Sunflower 199
phylogenetic analyses on 47 domesticated and wild germplasm accessions

using 122 SSR markers distributed throughout the sunflower genome.
These authors found extraordinary allelic diversity in the Native American
land races and wild populations, and progressively less allelic diversity in
germplasm produced by successive cycles of domestication and breeding,
suggesting that the contemporary oilseed sunflower pool could profit from
an infusion of novel alleles from the reservoir of latent genetic diversity
present in wild populations and Native American land races. Finally, Zhang
et al. (1995) used RFLPs to screen inbred lines for intra-line polymorphisms.
Although they found polymorphism within the four lines screened, these lines
presented good uniformity of morphological characters in the field. It was
concluded that the polymorphisms stemmed from residual heterozygosity or
outcrossing, and proposed using RFLPs for distinctness, uniformity, and
stability testing in sunflower.
Heterotic group modelling in sunflower using molecular tools has been
reported (Hongtrakul et al. 1997; Cheres et al. 2000), although it failed to reveal
clear heterotic groups. Cheres et al. (2000) estimated the correlation between
genetic distance, heterosis, and hybrid performance using AFLPs and coances-
tries, and found that genetic distance alone was a weak predictor of hybrid
performance in sunflower.
6.8.2.2 Molecular Mapping of Simply Inherited and Complex Traits

Oil Content
Oil content in sunflower is considered to be quantitatively inherited, and
depends on both the percentage of hull weight in relation to whole seed weight
and the concentration of oil in the kernel. Leon et al. (1995b) identified six QTL
associated with oil content with predominant additive gene action, which
accounted for 56% of the genetic variation. Two of these QTL were associated
to kernel oil percentage, two of them with kernel percentage, and two of them
with both components. Later studies reported the identification of three (Mes-
tries et al. 1998), six (Mokrani et al. 2002), and eight (Leon et al. 2003) QTL
associated to achene oil content, some of them consistently identified across
environments. Recently, Tang et al. (2006a) identified six QTL for achene oil
concentration on LG 1, 4, 9, 10, 16, and 17 in a RIL population developed from
the cross RHA280 (confectionery line) x RHA801 (oilseed line). The QTL
individually explained 3.1–22.5% and collectively explained 55.7% of the phe-
notypic variability for achene oil concentration. QTL on LG 10, 16, and 17 were
centered on the phenotypic loci B (apical branching), hyp (hypodermal pig-
ment), and P (phytomelanin pigment), respectively. Hajduch et al. (2007) using
a proteomic approach reported 77 protein spots differentially expressed in the
high oil line RHA 801 versus the low oil line RHA 280. Identification of 44 of
these proteins indicated that the two main processes affecting low or high oil
concentration in these lines were glycolysis and amino acid metabolism.
Oil Quality
Fatty Acids
The molecular basis of modified fatty acid profile in the seed oil of sunflower
has been studied through a QTL and a candidate gene approach. A number of
sunflower genes, coding for enzymes involved in the fatty acid biosynthetic
pathway in seeds, have been cloned and their polymorphism studied in culti-
vated sunflower. Hongtrakul et al. (1998a) reported the isolation of two stear-
oyl-acyl carrier protein (ACP) desaturase genes (SAD17 and SAD6) in
sunflower that were highly expressed in seeds. The SAD enzyme desaturates
stearoyl-ACP to oleoyl-ACP. Candidate gene and QTL analysis revealed the
co-location of a major QTL associated to stearic acid content in the high stearic
acid mutant CAS-3 (genotype es1es1es2es2) with a SAD17 gene. The SAD17A
locus was found to co-segregate with Es1 (Pérez-Vich et al. 2002b). Using
RFLP-AFLP linkage maps constructed from two different mapping popula-
tions derived from CAS-3, the SAD17A locus was mapped to LG 1, and it was
found to underlie the major QTL affecting the concentration of stearic acid.
This QTL explained about 80% of the phenotypic variance of this fatty acid
(Pérez-Vich et al. 2002b) and it was named st1-SAD17A. Other minor QTL
affecting stearic acid content, which mapped to LG 3 (st2.1), LG 7, and LG 13
(st2.3), were detected in that study, although none of them was consistent
enough to be considered as a strong candidate for the Es2 locus (Pérez-Vich
et al. 2002b).
Since the highly significant effect of the macromutation Es1 reduced the power
of the QTL analyses to identify QTL with smaller effects on stearic acid levels,
another mapping population in which stearic acid segregated independently of
Es1 was developed from the CAS-20 line (genotype Es1Es1es2es2) (Pérez-Vich
et al. 2004a). An RFLP-SSR genetic linkage map from this population allowed
the identification of three QTL affecting stearic acid, located on LG 3 (st2.1), LG
11 (st2.2), and LG 13 (st2.3). The three QTL collectively explained 43.6% of the
phenotypic variation. On the basis of positional information, QTL on LG 11 was
suggested to be a SAD6 locus (Pérez-Vich et al. 2004a).
Very high stearic acid content in the sunflower mutant line CAS-14 is deter-
mined by the Es3 gene (Pérez-Vich et al. 2006a). Using bulked segregant analysis,
Pérez-Vich et al. (2006b) mapped Es3 to LG 8 of the sunflower genetic map, and
identified SSR markers closely linked to this gene (Fig. 6.1). Ms11, one of the
genes determining nuclear male sterility in sunflower, was also mapped to LG 8 at
a genetic distance of 7.4 cM from Es3.
Marker studies related to high oleic acid content in sunflower began with
the identification of two RAPD makers linked to the Ol1 gene (Dehmer and
Friedt 1998b). Subsequent studies demonstrated that the Ol1 gene cosegre-
gates with a seed-specific oleoyl phosphatidyl-choline desaturase gene
(FAD2-1) that is strongly expressed in normal-type (low oleic) and weakly
expressed in mutant (high oleic) lines (Hongtrakul et al. 1998b; Lacombe and
Bervillé 2001; Martı́nez-Rivas et al. 2001). The Ol1-FAD2-1 locus mapped to
6 Sunflower 201
Fig. 6.1 Composite map of

LG 8 containing the Tph2,
Es3, and Ms11 genes
determining high gamma-
tocopherol content, very
high stearic acid content,
and nuclear male sterility,
respectively. The map was
constructed from the F2
mapping populations P21
CAS-14 (Pérez-Vich et al.
2006b) and CAS-12
IAST-540 (Garcı́a-Moreno
et al. 2006). The ORS and
CRT prefixes denote SSR
marker loci, and the ZVG
prefix denotes INDEL
marker loci. The cumulative
distances in centimorgans
are shown at the left of the
map
LG 14 (Pérez-Vich et al. 2002b) of the public sunflower genetic map, and was
found to underlie a major oleic acid QTL explaining 56% of the phenotypic
variance for this character (Pérez-Vich et al. 2002b). Schuppert et al. (2006a)
determined the physical structure of the FAD2-1 locus and developed poly-
morphic sequence-tagged-site (STS) DNA markers diagnostic for the Ol
mutation. Schuppert et al. (2005) indicated that the mechanism underlying
the Ol mutation was a FAD2-1 silencing by RNA interference.
Several studies have also been conducted to characterize modifying genes
affecting oleic acid content. Pérez-Vich et al. (2002b) described the existence of a
minor QTL on LG 8 which showed an epistatic interaction with the major QTL
for oleic acid at the FAD2-1 locus on LG 14. Lacombe et al. (2001, 2002)
identified a locus that suppressed the effect of the FAD2-1 locus, probably
through a mechanism of gene silencing. Schuppert et al. (2003, 2006b) described
the effect of at least seven genes from the fatty acid biosynthesis pathway,
including another oleate desaturase gene (FAD2-2) on LG 1, acting epistatically
with the Ol1-FAD2-1 locus on LG 14.
Tocopherols
Hass et al. (2006) and Garcı́a-Moreno et al. (2006) mapped the Tph2 gene
determining high gamma-tocopherol content in sunflower seeds to LG 8 of
the sunflower linkage map (Fig. 6.1). In addition, Hass et al. (2006) isolated and
characterized two paralogs of the gamma-tocopherol methyltransferase gene
(gamma-TMT-1 and gamma-TMT-2), that mapped to LG 8 and cosegregated
with the Thp2 locus. These authors also developed STS markers diagnostic for
Tph2. However, none of these DNA polymorphisms found between wild type
and mutant gamma-TMT-1 and gamma-TMT-2 alleles were associated with the
mutant phenotype, suggesting that the mutation may disrupt regulatory
sequences very tightly linked to the gamma-TMT locus (Hass et al. 2006).
Tang et al. (2006b) and Vera-Ruiz et al. (2006) mapped the Tph1 gene
controlling high beta-tocopherol content in sunflower seeds to LG 1. Tang
et al. (2006b) determined that the Tph1 mutation associated to the modified
tocopherol phenotype was a non-lethal knockout mutation in a 2-methyl-6-
phytyl-1,4-benzoquinone/2-methyl-6-solanyl-1,4-benzoquinone methyltrans-
ferase (MPBQ/MSBQ-MT) locus of LG 1 (MT-1) caused by the insertion of
a 5.2 kb Ty3/gypsy-like retrotransposon, and developed STS markers diagnos-
tic for wildtype and mutant MT-1 alleles. A second MPBQ/MSBQ-MT locus
mapping to LG 4 (MT-2) was also described to be associated to tocopherol
composition (Hass et al. 2006; Tang et al. 2006b). This locus was epistatic to the
MT-1 locus on LG 1 and had no effect in Tph1Tph1 or Tph1tph1 individuals,
but significantly increased beta-tocopherol content in thp1thp1 individuals.
Disease Resistance
Resistance to downy mildew is probably one of the best examples of the use of
major gene resistance. There are up to 10 resistance genes described, denoted Pl,
carrying resistance to various downy mildew races and mapped to genetic maps
(Vear 2004). Research on these genes has shown that there are at least three
clusters of genes plus several others that segregate independently from these
clusters. A cluster on LG 8 includes the Pl1, Pl2, Pl6 (from wild H. annuus), and
Pl7 (from H. praecox) genes covering a large area of about 0.5 cM with two
genetically distinct regions conferring resistance to different Plasmopara races
(Vear et al. 1997; Bouzidi et al. 2002). A second cluster on LG 13 includes the
Pl5 (from H. tuberosus) and Pl8 (from H. argophyllus) genes (Bert et al. 2001).
The Plarg (from H. argophyllus) gene was solely mapped to a telomeric region of
LG 1 (Dussle et al. 2004). Numerous resistance gene analogues (RGAs) have
been found clustered and linked to the Pl clusters on LG 8 (Gentzbittel et al.
1998; Gedil et al. 2001b; Bouzidi et al. 2002; Slabaugh et al. 2003) and LG 13
(Radwan et al. 2003), and to Plarg (Radwan et al. 2007).
Resistance to the parasitic weed Orobanche cumana appears to follow a
similar pattern to that of downy mildew. Dominant resistance genes Or1
through Or5, conferring resistance to races A through E, respectively, have
6 Sunflower 203
been described (Vrânceanu et al. 1980). The Or5 gene has been mapped to a
telomeric region of LG 3 of the sunflower genetic map (Lu et al. 2000; Tang
et al. 2003b; Pérez-Vich et al. 2004b). However, recent genetic and molecular
studies have revealed a more complex genetic control of broomrape resistance.
Pérez-Vich et al. (2004b) reported that phenotypic variance for race E resistance
was mainly explained by a major QTL on LG 3 (Or5 gene) associated to the
resistance or susceptibility character, while race F resistance was explained by
QTL with small to moderate effects, mainly associated with the number of
broomrapes per plant.
Lawson et al. (1998) developed SCAR markers linked to the R1 and Radv
genes conferring resistance to rust. Subsequent studies demonstrated that one
of the SCAR markers linked to the R1 gene mapped to LG 8 and was closely
linked to the downy mildew cluster on this LG (Slabaugh et al. 2003). Lenardon
et al. (2005) mapped the Rcmo-1 gene for resistance to sunflower chlorotic
mottle virus to LG 14.
Resistance to other diseases, such as Sclerotinia, Phomopsis stem canker,
and black stem is more complex, involving several loci with different effects
and highly dependent on environmental conditions. For this quantitative
resistance, there are no specific genes and races described, although lists of
QTL are becoming available. QTLs for resistance to Sclerotinia concerning
the capitulum reaction to the ascospore test have been identified on 14 of the
17 sunflower linkage groups in different crosses, explaining individually less
than 20% of the phenotypic variance (Bert et al. 2002, 2004; Yue et al. 2007).
One particular strong QTL was found on LG 8, linked to a protein-kinase
gene (Gentzbittel et al. 1998), but while it explained 50% of the variability in
one cross, in other crosses it explained only 15% or was not present. QTLs for
reaction to mycelium tests on leaves and capitula and for natural attack on
terminal buds have also been reported (Mestries et al. 1998; Bert et al. 2002,
2004), which often appear to co-localise with the QTLs for resistance to the
ascospore test (Vear 2004).
QTL studies on Sclerotinia midstalk rot resistance reported six to nine
QTL for each of the three resistance traits evaluated (leaf lesion, stem lesion,
and speed of fungal growth), each with a small effect. In total, between 24.4
and 33.7% of the genotypic variance for resistance against Sclerotinia could
be accounted for by these QTL (Micic et al. 2004). QTL for stem lesion
detected by these authors on LG 8 and 16 were demonstrated to be consistent
across generations (Micic et al. 2005a). Micic et al. (2005b) determined the
effect of three to four QTL associated to Sclerotinia resistance by selective
genotyping in a mapping population derived from crosses with a different
resistant line. In addition, these authors cross-referenced previous studies of
Mestries et al. (1998), Bert et al. (2002), and Rönicke et al. (2005) and found
that the same six linkage groups carried QTL for Sclerotinia resistance in
more than one population, and that LG 1, LG 9, and LG 10 had a significant
effect in the majority of the populations considered. Despite the complex
genetic architecture of Sclerotinia resistance, QTLs consistent across
environments (Bert et al. 2002), generations (Micic et al. 2005a), and mapping
populations (Rönicke et al. 2005; Micic et al. 2005b) have been identified,
which constitute valuable tools for the establishment of marker assisted
selection programs aimed at improving Sclerotinia resistance.
Bert et al. (2002) found three QTLs explaining up to 20% of the variability
for resistance reaction to natural attacks of Phomopsis stem canker, and other
three QTL for resistance reaction to artificial infections, one of them for both
types of infection. These authors also reported the co-localisation of a QTL
affecting resistance to Sclerotinia mycelium in leaves and another QTL for
resistance to Phomopsis in leaves, suggesting that these QTL could result
from the same components in the mechanism of resistance to these two faculta-
tive parasites.
In two independent studies, a significant number of QTL (four to seven) with
moderate effects on resistance to black stem were identified, explaining each
QTL from 5 to 17.5% of the phenotypic variance (Rachid Al-Chaarani et al.
2002; Bert et al. 2004). Subsequent studies on the population from Rachid
Al-Chaarani et al. (2002) testing different Phoma macdonaldii isolates allowed
the identification of a total of 27 resistance QTL for 4 isolates (Abou Alfadil
et al. 2007) and 10 resistance QTL for 2 isolates (Darvishzadeh et al. 2007), with
moderate individual effects ranging from 6 to 29%. Alignan et al. (2006), using
a cDNA microarray approach, identified several genes regulated in response to
Phoma macdonaldii. These authors proposed a model in which negative
regulation of a dual-specificity mitogen-activated protein kinase (MAPK)
phosphatase could be implicated in the defence mechanisms against this
pathogen via activation of a MAP kinase cascade that could trigger defence
responses such as thaumatin biosynthesis and phenylalanine ammonia lyase
(PAL) activation.
Developmental and Agronomic Traits

Male Sterility
Three out of 11 recessive genes controlling nuclear male sterility in sunflower
have been mapped. The Ms9 gene was mapped to LG 10 using TRAP and SSR
markers (Chen et al. 2006), whereas the Ms10 and Ms11 genes were mapped to
LG 11 and LG8, respectively using RFLP, SSR and INDEL markers (Pérez-
Vich et al. 2005).
Molecular studies have examined the nature of different CMS sources avail-
able in sunflower. Köhler et al. (1991) suggested that a new open reading frame,
orfH522, in the 30 -flanking region of the atpA gene was associated with the
CMS phenotype. Further studies using mtDNA genes and three probes for the
open reading frame clearly distinguished CMS sources by their mtDNA orga-
nization and CMS mechanism (Horn 2002). Kusterer et al. (2005) developed
PCR-based markers closely linked to the fertility restoration gene Rf1.
6 Sunflower 205
Self-incompatibility and Seed Dormancy

Wild populations of H. annuus are self-incompatible and have deep seed dor-
mancy, whereas modern sunflower cultivars are self-compatible and have short-
lived seed dormancy. Gandhi et al. (2005) mapped QTL for self-incompatibility
and seed dormancy in a backcross population from parents showing contrast-
ing characteristics for both traits. A single locus S for self-incompatibility was
identified and mapped to LG 17. The locus was tightly linked to a cluster of
QTL for several domestication and postdomestication traits. Three QTL for
seed dormancy with small individual effects in the predicted direction (wild
alleles decreased seed germination) were identified.
Embryogenesis
Plant regeneration by in vitro organogenesis offers the possibility of obtaining a
high number of regenerated shoots. Flores-Berrios et al. (2000) developed an
AFLP genetic linkage map from a RIL population exhibiting variability for
organogenesis traits. Six putative QTL for number of shoots per explant plated
and seven putative QTL for number of shoots per regenerating explant were
identified. For each trait, QTL explained 52 and 67%, respectively of the total
phenotypic variance.
Days to Flowering
Days to flowering is an important trait primarily controlled by the genotype,
photoperiod, and temperature. Few genetic factors for days to flowering have
been reported. Mestries et al. (1998) identified two QTL that accounted for 30%
of the phenotypic variation in a single environment. Leon et al. (2000) identified
five QTL that accounted for 73 and 89% of the phenotypic and genotypic
variations, respectively, across four locations with limited range of photoper-
iod. When evaluating the same population in locations with more different
photoperiods, Leon et al. (2001) found that the two QTL with the strongest
association with days to flowering were responsive to photoperiod.
Resistance to Abiotic Stresses

Poormohammad Kiani et al. (2007) conducted a pot experiment for mapping
water status traits and osmotic adjustment associated with drought tolerance.
The plants were induced to water deficit to compare QTL detection under well-
watered and water-stressed environments. In general, most of the QTL detected
under well-watered conditions were not detected in water-stressed conditions.
Eight QTL were detected for osmotic adjustment. The largest one, located at
LG 5 and accounting for 29% of the phenotypic variation, overlapped with
QTL for other water status traits such as leaf water potential, relative water
content, osmotic potential, and osmotic potential at full turgor.
Resistance to Herbicides
Sunflower biotypes resistant to two classes of acetohydroxyacid synthase
(AHAS)-inhibiting herbicides such as imidazolinones (IMIs) or sulfonylureas
(SUs) have been discovered. Kolkman et al. (2004) identified, cloned and
sequenced three AHAS sunflower genes: AHAS1, AHAS2, and AHAS3,
which were mapped to LG 9, 6, and 2, respectively. In addition, these authors
identified mutations in codons 197 and 205 in AHAS1 that conferred resistance
to IMI and SU herbicides, respectively, and developed a SNP genotyping assay
diagnostic for the codon 205 mutation.
6.8.2.3 Marker Assisted Selection

In contrast to the high number of reports on mapping of important traits
controlled by major genes and QTL, literature on practical application of
those markers in sunflower breeding programs remains very limited. There
are probably several scientific and logistical issues that must be still resolved
before practical marker assisted selection (MAS) strategies can flow from
mapping studies. Therefore in this section we will deal with factors determining
enhanced power of MAS and how they are faced in sunflower. Moreover, the
few examples of practical use of molecular markers in breeding programs
available so far will be highlighted.
MAS Optimization
Marker Validation and Refinement
Marker validation and refinement is one of the main factors enhancing selection
power of MAS. For markers associated to simply inherited traits, marker
validation and reduction of the distance between the marker and the gene of
interest is fairly straightforward. Examples of marker validation in different
genetic backgrounds have been reported for the Pl2 gene determining resistance
to different downy mildew races (Brahm et al. 2000), to the R1 and the Radv
genes conferring rust resistance (Lawson et al. 1998), and to the Or5 gene
conferring resistance to race E of broomrape (Tang et al. 2003b; Pérez-Vich
et al. 2004b). Improvement of marker accuracy for the Rf1 gene restoring pollen
fertility in PET1 based material was improved by using enlarged mapping
populations (Kusterer et al. 2005).
In many cases, the marker identified in the process of fine-mapping may not
be polymorphic in all the populations tested, thus requiring the identification of
alternative markers for those populations. Ideal markers for selection are those
based on gene mutations underlying the trait of interest. This kind of markers
has been developed in sunflower for oil quality traits and other simple traits.
For example, Tang et al. (2006b) determined that a non-lethal knockout muta-
tion in a MPBQ/MSBQ-MT locus on LG 1 (MT-1) was underlying
beta-tocopherol accumulation in sunflower seeds, and robust STS markers
6 Sunflower 207
diagnostic for wildtype and mutant MT-1 alleles were developed. Similarly,
Kolkman et al. (2004) identified a mutation in codon 205 in the acetohydrox-
yacid synthase gene AHAS-1 that confers resistance to imidazolinone (IMI)
herbicides, and developed a SNP genotyping assay diagnostic for it.
The situation becomes more complicated for QTL markers for complex
traits. Factors such as population structure and size, parental selection and
genetic background effects, epistasis, inaccurate phenotyping, or QTL x envir-
onment interactions contribute to bias the estimation of QTL effects, thus
reducing the likelihood of successful use of these QTL in MAS programs.
QTL validation in independent samples and in different genetic backgrounds
and environments is therefore necessary before using them in MAS breeding
programs. There are some good examples of QTL validation in sunflower,
making the validated QTL ideal targets for MAS. For Sclerotinia resistance,
QTL have been validated across environments (Bert et al. 2002), generations
(Micic et al. 2005a), and genetic backgrounds (Rönicke et al. 2005; Micic et al.
2005b). For oil content, QTL have been also validated across generations,
environments, and mapping populations (Leon et al. 2003; Tang et al. 2006a).
In addition to QTL validation, fine-mapping of QTL is very useful for
identifying tightly linked markers that will not suffer from loss of linkage due
to recombination between marker and QTL, and that will allow to minimize the
size to the introgressed fragment. The development of a high density sunflower
genetic map (one marker per 0.8 cM) through the mapping of 2,495 high-
throughput DNA marker loci (Knapp et al. 2007) will contribute to map
QTL with a high level of resolution. The development of specific genetic
resources such as near-isogenic lines (NILs), differing in a genomic segment
containing a target QTL, and RILs will also contribute to fine-mapping of
QTL. In sunflower, Micic et al. (2005a) re-estimated position and effects of a
number of QTL for Sclerotinia resistance in a RIL population developed from
F3 families where the QTL were originally identified. However, they only
obtained partial recovery of QTL detected in the earlier F3 generation in the
RILs for traits such as stem lesion. Pizarro et al. (2006) have developed QTL-
NILs varying in a target QTL for seed oil concentration, which allowed the
authors to determine its effect with higher resolution.
Association mapping has great potential for higher-throughput QTL detec-
tion. The method relies on linkage disequilibrium (LD) to study the relationship
between phenotypic variation and genetic polymorphism. The LD extent and
the application of association mapping in sunflower have not been studied in
depth. Recent reports by Liu and Burke (2006) and Kolkman et al. (2007), who
studied patterns of nucleotide diversity in genic loci from both wild and culti-
vated sunflower, demonstrated that SNP frequencies and LD decay were of
sufficient magnitude in wild populations (1 SNP/19.9 bp and LD decay within
200 bp), exotic germplasm accessions (1 SNP/38.8 bp and LD decay within
1,100 bp), and modern sunflower cultivars (1 SNP/45.7 bp and LD decay
within 5,500 bp) for high-resolution association mapping.
Assays Optimization and Cost Reduction

After the development of molecular markers and validation of their power for
selection for the trait, it is often necessary to optimize the assays, with driving
criteria being to reduce unit costs and turn around times while increasing
throughput and minimizing errors. Technologies that speed up the implemen-
tation process, reduce laboratory requirements or errors, and lower the cost
associated with scaling-up, are crucial to the success of MAS. One of the main
priorities included in the ‘‘White paper: Priorities for research, education and
extension in genomics, genetics and breeding of the Compositae’’ (The Com-
positae Genome project, http://compgenomics.ucdavis.edu/; 2007) for trans-
lating sunflower genomics into practical breeding programs was the reduction
of total marker costs. Advances in sunflower marker technologies have been
carried out in recent years. For SSR markers, PCR multiplexes for a genome-
wide framework of SSR marker loci developed by Tang et al. (2003a)
increased genotyping throughput and reduced reagent costs, which is essential
for repetitive genotyping applications. In addition, multicolour assays, SSR
primer design to facilitate ‘‘pooled amplicon multiplexing’’ by length in SSR
development, and SSR analyses in semi-automated, high-throughput geno-
typing systems (Yu et al. 2002; Tang et al. 2002) resulted in time-saving and
reduced costs for SSR assays. Currently, different techniques for SNP detec-
tion are being used in sunflower to type SNPs in a high-throughput, time-
saving and cost-effective fashion, including denaturing high-performance
liquid chromatography (DHPLC) (Lai et al. 2005), and single-base extension
or allele specific primer extension (Knapp et al. 2007).
Improved QTL detection methods that reduce costs have also been pro-
posed. Micic et al. (2005b) used selective genotyping for detecting QTL for
Sclerotinia resistance in sunflower. This method exploits the concentration of
most of the information for QTL effects in the ‘‘tails’’ of the quantitative trait
distributions. Accordingly, population sizes can be reduced to those individuals
found in these ‘‘tails’’. The authors concluded that selective genotyping can be
efficiently used for QTL detection and analysis of congruency for resistance
genes across populations, despite the limited sample size and the non-random
sampling used.
MAS in Sunflower Breeding Programs

The most common application of MAS is marker assisted/accelerated back-
cross breeding for gene introgression. Optimally, this is based on positive fore-
ground selection for donor trait, positive background selection for the recurrent
parent genome, and negative background selection against undesirable donor
parent alleles (Frisch et al. 1999). In general, marker assistance is expected to
provide higher efficiency, reduced cost, and/or shorter duration of the back-
cross breeding scheme, compared with conventional methods.
6 Sunflower 209
In sunflower, marker assisted backcross breeding is currently being carried

out in private breeding companies to accelerate the introgression of target genes
into elite germplasm. Traits such as downy mildew resistance, high oleic acid
content, and herbicide resistance are currently the main targets, although com-
plex traits such as resistance to Sclerotinia, Phoma and Phomopsis stem canker
are also taken into account. Despite there are no reports on such programs, it
seems that markers routinely used in plant cultivar development in private
programs are used mainly for selecting alleles with large effects on traits with
relatively simple inheritance. However, dissection of complex traits such as oil
content using molecular markers in sunflower is contributing to implement
MAS for such traits. For example, QTL associated to different components
that determine oil content (kernel percentage in the achene and kernel oil
percentage) have been identified and validated. Some of these QTL are asso-
ciated to the phenotypic loci B (apical branching), hyp (hypodermal pigment),
and P (phytomelanin pigment) (Leon et al. 1996, 2003; Tang et al. 2006a). This
fact was explained by Tang et al. (2006a) as a pleiotropic effect of such pheno-
typic loci on oil content, and allowed Leon et al. (1995a) to establish combined
marker and phenotypic (based on the hyp locus) assisted selection for high oil
content during the backcross process.
Codominant markers are most useful for marker-assisted backcrossing
because selection amonguy backcross progeny involves selection for heterozy-
gous progeny. If a dominant marker is used for selection, it will remain infor-
mative in subsequent backcross generations if the dominant allele (conferring
band presence) is linked to the donor parent allele. If the recessive allele is linked
to the donor parent allele, then progeny testing of each individual in each
backcross generation would be required, thereby doubling the number of gen-
erations required for backcrossing. Panković et al. (2007) proposed increasing
MAS efficiency in backcross programs to introgress the Pl6 gene conferring
resistance to downy mildew race 730 by using a combination of closely linked
codominant cleaved amplified polymorphic sequence (CAPS) markers with
dominant markers developed from resistance candidate genes.
Marker-assisted backcross breeding is also very effective in transferring
genes or QTLs determining valuable traits from wild donor genotypes into
elite breeding lines, reducing both the time required and the risk of undesirable
linkage drag with unfavourable donor attributes. To facilitate and accelerate
the introgression process of genes related to disease resistance, Slabaugh et al.
(2003) proposed the identification of allelic variation in wild species for specific
candidate genes such as RGAs, to identify potentially useful resistance genes
through disease screening, and to use markers developed from these RGAs to
track the gene in the introgression process. QTL and candidate gene analyses in
wild sunflower species is contributing to identify genes and QTL for adaptation
to salt or drought tolerance (Lexer et al. 2003a, b; Kane and Rieseberg 2007)
that could be exploited as a source of new genes to be introgressed into
cultivated sunflower. Despite the use of molecular markers to assist backcross
introgression of specific genes from wild species is still scarce, they have been
useful for the identification and characterization of interspecific hybrids (Natali

et al. 1998; Binsfeld et al. 2001).
Gene pyramiding is a useful approach to enhance the durability and degree
of pest and disease resistance, or to increase the level of abiotic stress tolerance.
Genes controlling resistance to different races or biotypes of a pest or pathogen
and genes contributing to agronomic or seed quality traits can be pyramided
together to maximize the benefit of MAS through simultaneous improvement
of several traits in an improved genetic background. Vear (2004) suggested that
major genes need to be backed up by quantitative, non-race specific resistance
QTL for increasing resistance durability. For this purpose, the use of molecular
markers is essential, since partial resistance conferred by these QTL can not be
determined phenotypically if combined with major resistance genes. Molecular
markers will be very useful to pyramid resistance genes tightly linked in clusters,
a virtual impossibility in practice using phenotypic analysis alone (Slabaugh
et al. 2003). For partial resistances such as Sclerotinia and Phomopsis stem
canker, a very important step towards the improvement of the level of resistance
is the use of MAS to combine different resistance QTL.
Different strategies are currently being carried out to enhance the efficiency
and scope of molecular breeding. The development of BAC (bacterial artificial
chromosome) and BIBAC (binary-bacterial artificial chromosome) libraries
(Gentzbittel et al. 2002; Özdemir et al. 2004; Feng et al. 2006; Tang et al.
2007) and linkage group-specific clones (Jan and Seiler 2007) is providing
resources and tools essential for comprehensive research of the sunflower
genome. These libraries are being used for isolating and physical mapping of
loci such as the FAD2-1 locus (Tang et al. 2007), or the fertility restorer Rf1
locus (Hamrit et al. 2006). In addition, combination of QTL mapping and gene
expression analysis and function elucidation is becoming an excellent tool for
dissecting QTL into Mendelian factors and improving the efficiency of mole-
cular breeding of complex traits in sunflower, such as drought tolerance (Poor-
mohammad Kiani et al. 2007).
6.8.3 Transgenic Breeding

Despite the increasing success of transgenic varieties of some major oilseed
crops such as soybean and canola, transgenic breeding research in sunflower
has been rather limited so far in comparison with the mentioned crops. A major
constraint for the advance of transgenic breeding have been the limitations of
the initial regeneration systems as well as problems in combining regeneration
and transformation within the same cells. Nevertheless, efficient transforma-
tion protocols with high reproducibility and high transformation frequency
have been developed (Mohamed et al. 2006).
Most of the development of transgenic varieties has been conducted by
seed companies, which have incorporated agronomically important traits.
6 Sunflower 211
The most important ones for which information is available are tolerance to
the herbicide glyphosate, by expression of 5-enolpyruvishikimate-3-phos-
phate synthase (EPSPS) genes from Agrobacterium tumefaciens, tolerance
to the herbicide glufosinate ammonium by expression of phosphinothricin
acetyltransferase (PAT) genes from Streptomyces spp., resistance to Lepidop-
tera by expression of Cry toxins genes from Bacillus thuringiensis (Bt genes),
resistance to Coleoptera by expression of trypsin inhibitor genes from cowpea
(Vigna unguiculata) plus lectin genes from snowdrop (Galanthus spp.), resis-
tance to Sclerotinia by expression of oxalate oxidase genes from wheat or
barley, and enhanced rubber production by expression of genes from guayule
(Parthenium argentatum) (Cantamuto and Poverene 2007).
Transgenic lines have also been produced at the public sector. Rousselin
et al. (2002) developed transgenic sunflower lines with reduced stearic acid
content by expression of a delta-9 stearoyl desaturase gene from castor bean
(Ricinus communis). Sawahel and Hagran (2006) produced transgenic plants
resistant to Sclerotinia by expression of the human lysozyme gene.
The risks associated with the gene flow from transgenic cultivars to the wild
flora are a matter of general controversy. In the case of sunflower, the risk is
particular high in North America, centre of origin for the genus, but also in
many other parts of the world where feral and naturalized populations of wild
Helianthus species are present (Bervillé et al. 2005). Examples of adaptative
advantages associated with the flow of transgenes to wild Helianthus popula-
tions have been reported by Snow et al. (2003) and Burke and Rieseberg (2003).
6.9 Seed Production

The sunflower breeder identifies inbred lines or open pollinated varieties that
have better performance than the currently used ones. Once the preliminary
trials suggest that an inbred line or open pollinated variety has potential, the
breeder increases the seed supplies and produces larger quantities of seed for
expanding testing. Seed at this stage is referred as ‘‘Breeder seed’’ because the
breeder is responsible for maintaining purity and increasing seed supplies of the
line or variety. When applied to hybrid varieties, it refers to the seed of male-
sterile, maintainer, and restorer lines. The initial increase of breeder seed is
known as ‘‘Foundation seed’’. It is handled to maintain specific genetic purity
and identity. ‘‘Registered seed’’ is the progeny of breeder and foundation seed
handled under procedures acceptable to the certifying agency to maintain
satisfactory genetic purity and identity. ‘‘Certified seed’’ is the progeny of
breeder and foundation seed handled to maintain satisfactory genetic purity
and identity which is approved by the certifying agency.
Hybrid seed is the first generation of seed of a cross produced by controlling
the pollination and by combining two or more lines. Single hybrid sunflower
seed is produced through the controlled crossing of male (restorer or R) and
female (male-sterile or A) lines. The A-line is maintained by crossing to a

genetically identical male-fertile line with a fertile cytoplasm, referred to as
maintainer or B-line. The commercial seed is usually grown from hybrid seed
and it is planted for any use except for seed production.
Increasing breeder seed and foundation seed of parental lines and certified
seed of hybrids is a time-consuming and critical operation in breeding programs
requiring full-time personnel to be in charge of operations. It requires systema-
tic planning and management on the part of seed producers. The production,
processing and marketing of the certified seed is exclusively the responsibility of
the seed producers. The seed-certifying agencies set up the procedures by which
each class of seed may be produced, the standards of purity and identity, and
also assume the responsibility for inspecting, sampling testing, and certifying
that the seed meets certification standards. Exact certification procedures vary
from country to country.
6.9.1 Maintenance and Increase of Parental Lines
Increases of A, B, and R lines are initially accomplished under bags in nurseries

to check for purity and stability of the cytoplasmic male sterility and to
eliminate off-types. These increases are often carried out under isolation in
screened cages to eliminate bagging. A hive of bees is placed inside each cage to
pollinate lines to be crossed and to eliminate hand crossing. An increase of the
R-line may not require pollination by bees. Usually lines are planted on differ-
ent dates so that these cages can be utilized for several increases each season.
For small increases, hand crossing of the A-line with the B-line may be done.
Hand pollination should be carried out in the morning on all days throughout
the flowering period.
Field increases of breeder/foundation seed of the female (A) line involves
planting the A and B lines using a ratio 1:1 or 2:1 (A:B) at low plant populations
in rows spaced 75–90 cm apart. The production field is isolated at least 6–8 km
from commercial fields or wild populations in countries such as USA where
those are frequent. One hive of bees per hectare is placed in the field for
pollination of the female parent.
An occasional problem in converting certain lines to cytoplasmic male
sterility is the occurrence of fertile plants in the progeny of crosses between
maintainer and male-sterile lines. The frequency of fertile plants was estimated
5.7% in a study involving 500 inbred lines (Vrânceanu and Stoenescu 1980).
Because of potential problems with fertile plants in increasing foundation seed
and in hybrid seed production, many breeders use the system of paired crosses
(Vrânceanu 1974) in converting and maintaining A- and B-lines. In this system,
the identity of individual A- and B-line plants is maintained and only those
B-line plants that produce completely sterile progeny are used for further
multiplication.
6 Sunflower 213
6.9.2 Commercial Hybrid Seed Production
6.9.2.1 Isolation
Similarly to the multiplication of parental lines, maintaining the recom-
mended isolation from other sunflower crops or wild species is a crucial
requirement in hybrid seed production to maintain genetically pure hybrid
seed. Pollen from external sources will contaminate the crop, causing tall
plants, reduced disease resistance, reduced yield potential, and in the case of
wild sunflowers, multi-headed plants. Seed companies try to avoid this carry-
ing out seed production in regions with no major commercial production to
eliminate the problem of isolation from cultivated sunflower. In the USA,
where wild sunflower is commonly present, wild plants must be removed from
the area of seed production before flowering to avoid undesirable cross-
pollination (Miller 1987). Considering the role of honeybees in pollination
and their flight range, seed certifying agencies have established minimum
isolation requirements of 1.6–4.8 km between seed production fields and
those of commercial sunflower in order to maintain the genetic purity of
parental lines or hybrids. Space isolation is the most important factor to be
considered for the production of quality seed. When space isolation is not
possible, time isolation of about 30 days is satisfactory. This means that the
flowering stage of the parental lines in the seed production field should be at
least 30 days earlier or later than that of other varieties grown within the area
to avoid contamination by pollen.
6.9.2.2 Plant Population and Planting Methods

Optimum plant population for hybrid seed production depends on the char-
acteristics of the female and male parents, environmental conditions, and
desired seed size. For most of the oilseed type hybrids, plant population varies
from 40,000 to 60,000 plants under irrigation. Optimum populations for
confectionary type hybrids are higher when smaller seed is desired for the
grower. However, when the market demands larger seeds, plant populations
of 40,000 plants and wider spacing between rows are used. The ratio of female
to male rows usually ranges from 2:1 to 6:1, depending on the pollination
ability of the male parent, similarity in the flowering dates of the male and
female parents, and number of row units used (Vrânceanu 1974). For exam-
ple, if the restorer line incorporates the recessive branching trait, which allows
the production of pollen during longer periods, the number of female rows can
be increased. One important consideration in planting the seed production
field is the staggered sowing of the parental lines in order to achieve flowering
synchronization between the female and R-line to avoid problems in hybrid
seed set.
6.9.2.3 Pollination
In the maintainer as well as in the hybrid seed production plots, pollination is a
crucial aspect to be considered. Hives of honeybees are placed in the hybrid seed
production field at the beginning of anthesis. The number of hives depends on
the plant population and stage of flowering. Seed producers generally use one to
four hives per hectare during the heaviest pollinating period. An adequate
number of hives is important since placing too many may force bees to forage
other sources of pollen and increase the percentage of outcrosses.
6.9.2.4 Roguing
Roguing is an essential practice in sunflower hybrid seed production for obtain-
ing physical and genetic purity. The objective of roguing is to remove before
anthesis the plants that do not meet the expected characteristics of the line (off-
types). It has to be strictly carried out in all the stages of crop growth.
A north–south orientation of the rows to improve its efficiency is recommended
(Miller 1987). In breeder/foundation seed as well as in certified seed production,
male-fertile plants within the male-sterile line are easily identified by dark
anthers and pollen production. In addition, plants with other morphological
deviations have to be removed in A- and B-lines before flowering. In breeder
and foundation seed production of restorer lines as well as in certified seed when
R-lines are involved, off-types have to be removed before anthesis. A row
spacing of 76–90 cm facilitates walking between rows for observing plants.
Twin rows, consisting of two rows of the female parent planted 60 cm apart
and 90 cm from the next twin-row set, have also been used.
6.9.2.5 Harvesting and Processing

Processing hybrid seed from the field to the bag requires many operations.
Harvesting seed production fields is a critical operation. It should not start until
moisture has reached 11–13%, and much care must be taken to prevent exces-
sive damage to the achenes. Rows of the male parent generally are removed
before the female rows are harvested to avoid contamination. In some cases,
seed producers remove rows of the male parent after pollination. After harvest-
ing, the seed is transported to the seed processing plant where it is cleaned,
graded, treated, and bagged. Quality control is required in order to maintain the
seed quality certification standards. Seed is stored under adequate conditions of
temperature and relative humidity to avoid deterioration of quality. Commer-
cial seed lots are generally treated with a combination of insecticides and
fungicides conferring protection against damage and stand loss by early season
soil and foliar insects as well as early season diseases, particularly downy
mildew.
6 Sunflower 215
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Chapter 7
Flax
Scott D. Duguid
7.1 Production and Utilization
Over the centuries the production of flax (linseed, Linum usitatissimum L.) has
spread across Europe, Africa and finally North America where it was the first
oilseed to be widely grown in western Canada. Today, world flaxseed production
has ranged between 2.0 and 3.0 million tonnes over the last 10 years. In
2006–2007, the production of flaxseed was 2.7 million tonnes, which represents
about 0.7% of the world production of oilseeds. Canada is the world’s largest
producer representing 40% of world production and most of the export trade.
China, the United States of America and India together account for 40% of the
world production. Within the European Union, the main producers of flaxseed
are Germany, United Kingdom, and France. Production has remained relatively
stable in China, India, and the European Union over the last decade (Table 7.1).
Primarily, flax is grown for seed with fibre production as a by-product.
Today, the unique properties of flaxseed with high levels of alpha linolenic
acid (ALA) in the oil differentiate it from other oilseeds in the industrial, human
food and animal feed market. Industrially, the oil is a major ingredient in
linoleum flooring and is also used in paints, stains and surface coatings as
well as in the oleo-chemical industry due to its rapid oxidation properties.
Technological developments since the 1950s, such as increased usage of latex
paints and petroleum based floor covering has reduced industrial demand.
Since the turn of the century, the trends to green and health oriented products
have resulted in new opportunities for flax, for instance the non-allergenic and
biodegradable characteristics of linoleum, along with quality improvements
have led to a resurgence in demand for linoleum flooring.
The use of flaxseed as a functional food has been traditional in many parts of
the world such as Northern Europe, however, the functional properties of flax
have only recently received considerable attention in North America due to the
S.D. Duguid (*)

Morden Research Station, Agriculture and Agri-Food Canada, Morden, MB,
Canada R6M1Y5
e-mail: scott.duguid@agr.gc.ca

234 S.D. Duguid
Table 7.1 World flaxseed supply and disposition

2006–2007
2005–2006 (estimated) 2007–2008 (forecast)
Million tonnes
Carry-in stocks 0.09 0.53 0.70
Production
Canada 1.08 1.04 0.60
China 0.48 0.48 0.45
United States 0.48 0.35 0.23
India 0.23 0.21 0.21
EU 0.18 0.18 0.17
C.I.S. 0.10 0.10 0.10
Bangladesh 0.05 0.05 0.05
Argentina 0.05 0.05 0.04
Other 0.20 0.21 0.28
Total production 2.85 2.67 2.13
Total supply 2.94 3.20 2.83
Crush 2.09 2.19 2.15
Other 0.32 0.31 0.35
Total use 2.41 2.50 2.50
Carry-out stocks 0.53 0.70 0.33
Trade 0.80 0.86 0.86
Source: Bi-weekly Bulletin – Flaxseed Situation and Outlook, Agriculture and Agri-Food
Canada.
numerous health benefits attributed to this oilseed. Flax is classified as a

functional food or nutraceutical product primarily because of the presence of
significant quantities of three of the most physiologically-active components:
(1) the omega-3 essential fatty acid, alpha linolenic acid, (2) lignans, a major
class of phytoestrogens and (3) soluble fibre primarily in the form of mucilage.
Benefits from flaxseed can be introduced to the diet through flaxseed oil, whole
or milled flaxseed, or through many products such as omega-3 eggs produced
by hens on flaxseed-fortified rations or in products that are readily available in
supermarkets and that contain flaxseed include breads, cereals, crackers, energy
bars, baking mixes, snacks, soups and waffles.
Flaxseed oil and meal have for many years been recognized as valuable
components in animal nutrition. The animal feed market offers significant
opportunities for flax to: (1) improve animal productivity as an integral com-
ponent of the feed and (2) to develop healthier food applications for humans
from flax fed animals. The benefits of flaxseed as animal feed include improving
growth and lessening disease and stress related conditions. For poultry, flaxseed
in the laying hens’ rations results in eggs that are higher in omega-3 fatty acid,
which health conscious consumers want in their diet. For swine, the inclusion of
flaxseed in the diet not only changes the nutritional quality of the pork by
making more omega-3 fatty acid available to the consumer, but studies also
7 Flax 235
suggest that flaxseed in the diet of breeding sows produce larger and healthier
piglets. Flaxseed is routinely included in premium pet foods to improve the
overall health and appearance of cats and dogs. Research continues on how
flaxseed could be incorporated into rations for dairy cattle to create products
such as omega-3 enriched milk and cheese. In general, flaxseed is milled before it
is included in animal rations, to ensure maximum absorption of the nutrients in
the flaxseed.
Flax straw contains long stem fibres with unique properties that make it
useful in the production of fibre products for textiles but it is also used in the
paper and pulp industry as a pulp sweetener to strengthen recycled paper,
geotextiles, absorbent products, insulation. New developments are focusing
on using flax straw as an alternative fuel, which has a per tonne heating value
similar to soft coal and appears to be a carbon neutral method of producing
heat.
7.2 Origin and Taxonomy
The flax family Linaceae is geographically widespread with about 300 species
worldwide. Linum is the largest genus within the family with three geographic
centers of diversification: (1) the Mediterranean area; (2) southern North
America and all of Mexico; and (3) South America as well as in other areas of
Europe, Asia and the Americas. Several proposals for dividing the genus into
sections exist (Diederichsen and Richards 2003) and the status of many species
remains to be clarified. The diversity of species in the genus Linum differs in
various parts of the world. Chennaveeraiah and Joshi (1983) conducted cyto-
logical studies on 19 species and proposed phylogenetic relationships based on
chromosome numbers and similarities. Within the genus Linum, the chromo-
some number varies between 2n = 16 and 2n = 60, with most of the species
having either 2n = 18 or 2n = 30 chromosomes. Gill (1987) presented chromo-
some numbers of 41 species of the genus ranging from 2n = 16 to 2n = 80.
Further clarification to the phylogenetic relationships within the genus is
required as the systems proposed are artifical, in nature. Cross hybridization
between cultivated and wild species has found that interspecific hybridization
between cultivated flax and Linum angustifolium have been successfully con-
ducted. In addition Gill and Yermanos (1967) reported successful hybridization
with 3 different Linum species. However, it should be pointed out that none of
these interspecific crosses has had any practical use in flax breeding. Plant
systematics attempts to establish a system reflecting the evolutionary relation-
ships among plant species. As Diederichsen and Richards (2003) suggested,
natural systems in plant taxonomy are supportive for plant breeding because
they facilitate the targeted search for desirable traits of economic importance in
closely related species. The challenge for plant breeders is to introduce the
desired traits from the wild species (Table 7.2).
236 S.D. Duguid
Table 7.2 Distribution of species in the genus Linum for different areas of the world
Area Number of species
Northern America and Mexico 63
Former Soviet Union 45
Turkey 38
Europe 36
France 24
Italy 20
Bulgaria 19
Morocco 19
Lebanon and Syria 17
Algeria 15
Iran 13
Germany 10
Portugal 10
USA, North-East 10
Central Asia 9
Canada 8
Switzerland 8
Afghanistan 7
Alava/Spain 7
Mallorca/Spain 6
Australia 6
Libya 6
Pakistan, West 6
Cyprus 5
India 5
Egypt 4
Tropical East Africa 4
Belgium 3
Iraq 3
Arabia 2
Japan 2
Scandinavia 2
Source: Diederichsen and Richards (2003).
7.3 Variety Development

Cultivar development for Canada and the USA has been largely a task of public
breeding programs. Four major breeding programs develop flax varieties for
Canada and the USA: Agriculture and Agri-Food Canada program located at
the Morden Research Station in Morden, Manitoba; Crop Development
Centre program located at the University of Saskatchewan in Saskatoon,
Saskatchewan and the Viterra program located at the Alberta Research Council
site at Vegreville, Alberta; and finally the North Dakota State University
program at Fargo, North Dakota, USA.
7 Flax 237
Since the early 1900s, Agriculture and Agri-Food Canada and its predeces-
sors have been active in the development of new flax varieties for Canada, and,
in particular, for the Canadian prairies. The initial program at the Central
Experimental Farm in Ottawa produced varieties such as Diadem Ottawa
770B, Ottawa 829C, and Novelty. During the 1950s, this program was particu-
larly active, releasing varieties such as Linott, Raja and Rocket. The 1950s and
1960s also marked the beginning of an evolution and transition in flax breeding
in Canada. A new program was initiated at the Indian Head Experimental
Farm and the Winnipeg Cereal Breeding Laboratory which led to the develop-
ment of the variety Cree. In the 1960s, a breeding program was also conducted
in Alberta at the Fort Vermillion Experimental Farm and Beaverlodge
Research Station, producing the variety Noralta, the predominant variety
grown in northern Alberta and Saskatchewan. The breeding programs of
Agriculture and Agri-Food Canada were moved and consolidated to Winnipeg
in 1960; then moved to Morden, Manitoba, where they still exist. The varieties
Dufferin, McGregor NorLin, NorMan, AC Linora, AC McDuff, AC Emerson,
AC Carnduff, Lightning, Hanley, Macbeth, Prairie Blue, Prairie Thunder, and
Prairie Grande have been released by Agriculture and Agri-Food Canada
(Table 7.3).
A modest breeding program was carried out at the University of Sas-
katchewan from the 1920s to the 1960s, which produced the varieties
Royal and Redwood 65. The program was enlarged in 1974 when the
Table 7.3 North American flax cultivars released since 1990

Year of Breeding
Cultivar Pedigree release program Reference
AC Linora Linott/NorMan 1991 Agriculture and Kenaschuk
Agri-Food and Rashid
Canada (1993)
Neche CI2847/Culbert 79 1991 North Dakota Hammond
State et al. (1991)
University
AC McDuff FP766/FP775 1992 Agriculture and Kenaschuk
Canada (1994)
Omega CI3036/Flor 1992 North Dakota Miller et al.
State (1992)
University
Verne Culbert/Hioil 5017 1992 Minnesota Comstock and
Agriculture Putnam
Experiment (1992)
Station
LinolaTM 947 McGregor/Zero// 1993 Viterra Dribnenki and
84495/3/McGregor2 Green
(1995)
238 S.D. Duguid

Year of Breeding
AC Emerson Noralta/Vimy 1994 Agriculture and Kenaschuk
Canada (1996)
Prompt BFP/Culbert 1994 South Dakota Grady and
State Lay (1994)
University
Day N707//CI2777/N419 1994 South Dakota Grady and
State Lay (1994)
University
LinolaTM 989 McGregor/Zero// 1995 Viterra Dribnenki
CPI8495/3/ et al. (1996)
McGregor3
CDC Tissue Culture 1995 Crop Rowland et al.
Normandy Derived Development (2002)
Somaclonal Variant Centre
of McGregor
CDC Triffid Sulfonylurea 1996 Crop McHughen
Herbicide Resistant Development et al. (1997)
– Transgenic Centre
Never produced
commercially
CDC Valour NorLin/Vimy 1996 Crop Rowland et al.
Development (2002)
Centre
AC Watson NorLin//FP775/ 1997 Agriculture and Kenaschuk
CI2941 Agri-Food and Rashid
Canada (1998)
AC Carnduff Dufferin//NorLin/ 1998 Agriculture and Kenaschuk
Culbert 79 Agri-Food and Rashid
Canada (1999)
CDC Arras Vimy/FP833 1998 Crop Rowland et al.
Development (2002)
Centre
CDC Bethune NorMan/FP857 1998 Crop Rowland et al.
Development (2002)
Centre
Taurus Cebeco 3100 1999 Cebeco Zaden
B. V.,
Netherlands
Cathay M23/CI2932 1998 North Dakota
State
University
Pembina FP805/SD8308 1998 North Dakota
State
University
7 Flax 239

Year of Breeding
TM
Linola 1084 Flanders/FP946 1999 Viterra Dribnenki
et al. (1999)
LinolaTM 2047 989//Windemere/ 2000 Viterra Dribnenki
M2702 et al. (2003)
Hanley Flanders/AC 2001 Agriculture and Duguid, S. D.
Emerson Agri-Food et al. (2003)
Canada
Lightning AC McDuff/AC 2001 Agriculture and Duguid, S. D.
Watson Agri-Food et al. (2003)
Canada
CDC Mons Flanders/FP926 2002 Crop Rowland et al.
Development (2003)
Centre
Macbeth M2701/AC Linora 2002 Agriculture and Duguid, S. D.
Agri-Food et al. (2003)
Canada
Prairie Blue Flanders/FP956 2003 Agriculture and Duguid et al.
Agri-Food (2004)
Canada
CDC Gold ED-48/YSED-19 2003 Crop
Development
Centre
LinolaTM 2090 92-7335/92-7337 2003 Viterra Dribnenki
et al. (2004)
LinolaTM 2126 SP992/94-7889 2004 Viterra Dribnenki
et al. (2005)
Nekoma U605/Bison 2004 North Dakota Hammond
State et al. (2004)
University
York U23/CI2929// 2004 North Dakota Hammond
McGregor State et al. (2004)
University
CDC Sorrel FP956/Vimy 2005 Crop
Development
Centre
LinolaTM 2149 SP1084/96-32-F3 2005 Viterra Dribnenki
et al. (2007)
Prairie Thunder FP974/FP1043 2006 Agriculture and
Agri-Food
Canada
Prairie Grande AC Watson/CI3395 2007 Agriculture and
Agri-Food
Canada
240 S.D. Duguid
Crop Development Centre (CDC) initiated a flax breeding program. It has

since produced the cultivars Vimy, Somme, Flanders, CDC Normandy,
CDC Valour, CDC Arras, CDC Bethune, CDC Mons, and CDC Sorrel as
well as the solin variety CDC Gold. Other varieties produced at the Crop
Development Centre include Andro (tissue culture derived) and CDC
Triffid (first transgenic flax cultivar – never commercially produced).
Both of these varieties have now been deregistered and are not commer-
cially available (Table 7.3).
In 1987, a solin (low alpha linolenic acid) breeding program was
initiated by Biotechnica Canada in cooperation with Australia’s Common-
wealth Scientific & Industrial Research Organization to develop the low
alpha linolenic acid flax, subsequently known as solin utilizing a double
mutation. In 1990, UGG Ltd. (now known as Viterra) purchased Biotech-
nica’s interest in the program and moved the program from Calgary to the
AAFC research station located at Morden, Manitoba and the research and
evaluation farm at Rosebank, Manitoba. In 2005, the program moved to
the Alberta Research Council research facility at Vegerville, Alberta. This
breeding program has produced the solin cultivars LinolaTM 947, 989,
1084, 2047, and 2090 (Table 7.3).
Over the last number years the flax breeding has decreased significantly
in the United States of America with the only remaining active program
being located at North Dakota State University. In 1984, with the retire-
ment of breeder at the Minnesota Agriculture Experiment Station, the
program was terminated and 1997 was the last year that material was
evaluated in South Dakota State University program. The USDA/ARS
flax improvement research at North Dakota State University was reduced
to maintenance of the Flax World Collection and coordination of regional
testing several years ago. Several years ago the collection was moved to
Ames, Iowa (Table 7.3).
Developing improved flax cultivars is a continuing process using the
germplasm base, created by the cumulative efforts of flax workers over
many years. Each improvement in germplasm represents an increment
added to the flax gene pool. Many lines from the Canadian and USDA
germplasm collections have provided valuable sources of variation that
were successfully used to develop improved cultivars. Valuable germplasm
lines that researchers developed are freely exchanged among flax research-
ers, which increases the diversity of the genetic base of gene pools of each
program. Continued progress in the improvement of yield, quality and
disease resistance demonstrated by a succession of cultivars in all flax
growing areas suggest further progress can be anticipated. In addition,
identification of genetic variation for useful, unique characteristics of flax
may provide a germplasm base for developing cultivars suitable for new
uses for the flax crop.
7 Flax 241
7.4 Variety Development Objectives
Breeding objectives vary among flax breeding programs depending upon the parti-
cular problems that exist in the specific production area. Objectives that are com-
mon to most flax breeding programs are high yield, suitable maturity, lodging
resistance, good grain quality and disease resistance. Some problems may be unique
to a specific area and of little concern in other areas. For example, breeding for
chlorosis resistance is an important objective in Manitoba but of little consequence
in Saskatchewan and Alberta where the soil is not as calcareous as in Manitoba.
7.4.1 Yield
Breeding for high grain yield is clearly the most important objective for flax
improvement. Over the past century, flax breeders have been extremely
successful in exploiting the germplasm base to produce the currently avail-
able cultivars but if flax is going to compete with other oilseeds for acreage
then substantial improvements in yield will be necessary. The primary yield
components of flax are: (1) the number of bolls per unit area, (2) the
number of seeds per boll, and (3) seed weight. Fortunately, flax has the
capacity of compensation among the yield components. Although it is
possible to breed for high yield per se, the flax breeder must select for
other qualities such as disease resistance, lodging resistance, and maturity
adaptation to develop cultivars that consistently produce high yields.
7.4.2 Maturity
Flax is a cool season crop, so maturity suitable for the intended production area
is important. The development of an early maturing variety with specific
adaptation to northern conditions and that matures in about 90 days instead
of 100 days should result in yield increases. There has been a tendency over the
years to breed flax for early maturity. The reasoning behind is that early
maturing versus late maturing varieties have a better opportunity to escape
damage or stress from abiotic stresses such as heat and drought, cold, frost and
diseases. Flax maturity is highly heritable and available germplasm is adequate
for developing cultivars with maturity suitable for the intended area of
production.
7.4.3 Lodging Resistance

Lodging caused by severe storms, high soil fertility or delayed harvest
frequently causes serious flax losses and grain quality reductions particularly
242 S.D. Duguid
if it occurs early during grain filling. Severely lodged flax is also more suscep-
tible to diseases such as pasmo and sclerotinia. Lodging resistance helps to
ensure good grain filling and minimal harvest losses. In 1993, 80% of the flax
acreage in Manitoba was grown to medium-early maturing varieties, predomi-
nantly NorLin, NorMan and Somme. These varieties are more susceptible to
lodging than the late maturing varieties such as McGregor and AC McDuff.
Yield losses in flax resulting from lodging have not been well documented, and
yields of susceptible variety Vimy in Manitoba are often more than 50% of the
yields of other varieties of similar maturity in cooperative tests grown under
severe lodging conditioning.
Grain yield losses caused by lodging in small plot breeding trials generally
are not indicative of losses that occur under practical conditions on the farm,
and consequently, some breeders may underestimate the importance of lod-
ging resistance especially if wet conditions occur for several days after lodging
did. Flax breeders certainly have been aware of the need for better lodging
resistance in flax cultivars, but there have been few programs where improve-
ment of that trait was the top priority for the following reasons: (1) developing
cultivars with resistance to major diseases has required a major portion of the
limited resources available for flax improvement, (2) heritability of lodging
resistance is relatively low, (3) the trait is difficult to measure because of
undependable, highly variable occurrence of stresses causing lodging, and
(4) combining lodging resistance with high yield and good seed quality is
difficult.
7.4.4 Grain Quality

Grain quality factors such as high oil content and desirable fatty acid
composition and high protein content have received increased attention
from flax breeders in recent years. The important grain quality factors are
under genetic control. There is sufficient genetic diversity in the cultivated
and germplasm collections to ensure continued improvements of most
quality traits.
7.4.4.1 Oil and Fatty Acid Composition

The most important quality parameter for flax is high oil content. Over the past
10 years, Canadian flaxseed contained, on average 44% oil (Table 7.4). Oil
content on individual farm samples can vary by as much as 15% with a range
of 35–50% being reported for farm grown samples tested over the past 5 years.
As the seed from individual farm samples moves through the handling system, it
is combined and the range of oil content decreases so that the range of oil content
in Canadian exports of flaxseed has only been 3.5%, from 42 to 46%. Our
7 Flax 243
Table 7.4 Flaxseed, No 1. Canada Western Seed Quality for 1997–2006 harvest survey
Protein Fatty acid, % in Oil Free
Oil content content Iodine value fatty
Year % % Wijs Palmitic Stearic Oleic Linoleic Linolenic acid, %
1997 43.9 23.5 193 58.0 0.20
1998 43.6 22.9 190 5.5 3.6 19.4 14.3 56.8 0.19
1999 43.9 21.8 196 5.4 3.1 17.1 14.7 59.6 0.17
2000 44.1 22.4 194 5.4 3.2 17.9 14.2 58.9 0.26
2001 44.1 24.1 190 5.2 3.7 19.5 15.1 56.3 0.40
2002 45.5 23.7 195 4.9 3.1 17.3 15.1 58.9 0.29
2003 44.2 25.6 184 5.2 3.7 22.4 15.0 52.9 0.15
2004 44.8 22.1 201 4.9 3.0 14.5 15.8 61.6 0.26
2005 46.2 22.0 194 5.0 3.3 16.8 16.3 57.7 0.18
2006 45.9 23.6 190 5.0 3.6 19.5 15.6 55.8 0.16
2007 44.7 24.3 184 5.0 3.6 20.6 16.2 52.6 0.16
1997–2006 44.7 23.2 193 5.2 3.4 18.2 15.1 57.7 0.22
Source: Canadian Grain Commission.
highest oil producing variety at present is AC McDuff with an oil content of

48%. Recently, Agriculture and Agri-Food Canada received a recommendation
to register FP2188 which is the first flax variety with a oil content of 50% of
which 59% is alpha linolenic acid.
The nutritional quality of a vegetable oil for human and animal consumption
or as industrial oil are determined by its fatty acid composition. Flax oil is
characterized by high alpha-linolenic acid contents (>50%) and moderate
concentrations of oleic (approximately, 18%) and linoleic acid (approximately,
14%). Palmitic (about, 5%) and stearic (about, 3%) fatty acids are also present
(Tables 7.4 and 7.5). Flax oil is one of the most unsaturated common plant oils,
and its iodine value is usually greater than 185 Wijs units. The level of unsatura-
tion varies with both variety and environment. The level of linolenic acid over
the last 10 years (1997–2006) from Canadian flax according to the Canadian
Table 7.5 Flaxseed, No 1. Canada Western Solin Seed Quality for 1998–2005 Harvest survey
Oil Fatty acid, % in oil
content Protein Iodine
Year % content % value Wijs Palmitic Stearic Oleic Linoleic Linolenic
1998 42.8 23.3 140 6.4 4.1 16.2 70.0 1.9
1999 43.5 21.7 143 6.1 3.5 14.6 72.2 2.2
2000 44.6 22.5 143 5.7 3.6 15.4 72.0 2.1
2001 44.8 22.3 141 5.5 4.2 17.5 69.7 2.0
2002 46.2 22.7 144 5.3 3.5 15.7. 72.6 2.1
2003 46.4 26.0 139 5.9 4.0 18.3 68.3 1.8
2004 48.0 23.4 148 5.5 2.8 12.8 75.2 2.4
2005 49.1 22.0 145 5.7 3.2 14.4 73.3 2.2
1995–2004 45.1 22.9 143 5.9 3.7 15.8 71.2 2.0
Source: Canadian Grain Commission.
244 S.D. Duguid
Grain Commission was 57.7% with an iodine value of 192.7 Wijs. Environ-
mental studies have demonstrated that flax grown under cool conditions has
higher levels of linolenic acid and overall iodine value.
The need for various fatty acid modifications in flax is well established
and research efforts are underway to develop germplasm with the various
fatty acid profiles for use by the world flax industry whether as a func-
tional food, pharmaceutical or industrial uses because of their improved
biodegradability. Various sources of higher levels of linolenic acid are
available in flax and these are being introgressed into elite germplasm.
Lines with approximately 70% ALA have also been developed but require
agronomic improvement. Flax breeders, using mutation selection processes
have developed lines of flax with low levels of alpha linolenic acid (< 3%)
which have been given the common name solin to differentiate them from
traditional flax varieties. Examples of solin varieties include Linola 947,
989, 1084, 2047, 2090, 2126 and CDC Gold (Tables 7.3 and 7.5).
7.4.4.2 Protein
Historically, the flaxseed crush in Europe was driven by the demand for
linseed oil to be used in the production of linoleum, paints and other
industrial products. Recently, however, the demand for non-genetically
modified high protein meal (45–50%) is driving the crush and the produc-
tion of linseed oil. The linseed meal is fed to livestock, primarily in
Western Europe, while surplus linseed oil is sold to distant markets such
as China and North Africa. In general, linseed meal (1.2 Mt in 2001–2002)
is consumed in the country in which it is produced, but only 82,000 t were
exported from the producing country.
As with other oilseeds, there is an inverse relationship between oil and
protein in flaxseed. The relationship for flaxseed however, is not as strong
as for other oilseeds such as canola and, unlike in canola, the meal protein
is not related to the oil content. Seed proteins in flax consist of about 20%
albumins and 80% legumin-like proteins and are more liophilic than
soybean proteins. The legumin-like fraction is rich in sulphur amino
acids. Over the past 5 years, Canadian flaxseed has been found to contain
about 23% crude protein that translates into a meal protein content of
43–46% (Tables 7.4 and 7.5). On individual farm samples, protein ranged
from 17 to 29% while on exported flaxseed the range was 20.9–25.1%.
Various sources of higher protein content are available in flax and these
are being introgressed into elite germplasm. FP2188, the recently recom-
mended line for registration for Agriculture and Agri-Food Canada, is an
example of one of these sources of higher protein content (47%) in the
meal but also breaking the inverse relationship between oil and protein
content.
7 Flax 245
7.4.4.3 Mucilage
In flax, sugars and starch (digestable carbohydrates) are minimal to none whereas
the majority of carbohydrates are resistant to the action of digestive enzymes.
Flaxseed mucilage is an excellent source of dietary fiber and due to its highly viscous
nature also shows potential for being used as a food gum. Flaxseed mucilage is a
complex mixture of two polysaccharides, which differ in physiochemical properties
such as composition, molecular size, structural conformation and rheological
properties. Genetic variability exists for the content (4–7%) and physiochemical
properties of flaxseed mucilage (Diederichsen et al. 2006). This variability would
allow the development of cultivars that contain mucilage with rheological and
functional characteristics for specific end uses.
7.4.4.4 Lignan
There has been considerable interest in the inclusion of flaxseed in western diets
or as an isolated and purified compound for improvement of human health.
Part of this interest comes from studies indicating that there may be a significant
beneficial effect resulting from the biological activity of the lignan secoisolar-
iciresinol diglucoside (SDG). Although the level of SDG found in Canadian
cultivars is significant (13 mg/g for Flanders – 22 mg/g Linola 947 of the seed),
an increase in this level would be beneficial. Of the six major groupings of flax,
the Indian, the Mediterranean and Spring collections contain accessions with
the highest SDG levels, while Winter, Fiber and Forage types contain lower
levels of SDG on average. If a variety was developed with a meal SDG content
of approximately 40 mg/g, it would represent more than double the SDG
content of commercial flax meal which is typically in the 1.2–1.7% range.
7.4.4.5 Anti-nutritionals
Characteristics that limit utilization of flaxseed meal include the presence of
anti-nutritional factors such as cyanogenic glucosides and cadmium. As in the
case of canola, a leading factor in international competitiveness would be the
development of improved varieties low in anti-nutritional factors such as cya-
nogenic glucosides and cadmium. Hydrolysis of cyanogenic glucosides can
produce hydrogen cyanide, a potent respiratory inhibitor, and thereby limiting
the quantity (8–10%) of flaxseed that can be used in food products or feed
rations. Lowering of cyanogenic glucosides may also increase the value of other
value added products in the food and pharmaceutical markets such as proteins,
gums and lignans. Typically, varieties such as AC McDuff have a total level of
467 mg/100 mg of seed (390 mg/100 mg linustatin and 70 mg/100 mg of
neolinustatin). Accessions have been identified with lower levels of cyanogenic
glucosides and crossed with adaptable material. These crosses are beginning to
reach the yield trial level and have a 60% reduction in total cyanogenic
glucosides.
246 S.D. Duguid
Cadmium content in flaxseed is influenced by geography, soil type, fertilizer

usage, etc. All current varieties are high cadmium accumulators (range 1.3–2.0 ppm)
(Oomah et al. 2007; Grant et al. 2008). Flax is currently receiving increasing
attention from health professionals as a possible food supplement in human diets,
especially because of the evidence of its substantial health benefits. This will open the
way for acceptance of flax as a high quality source of improved protein meal and
value added products. The current limitations can be removed by improving the
quality of flaxseed meal through applied plant breeding programs. Accessions have
been identified with lower levels of cadmium uptake and crossed with adaptable
material. However, the lack of an inexpensive technique to evaluate the level of
cadmium has slowed the effort. With the development of an inexpensive testing
along with traditional breeding and double haploid techniques it should be possible
to develop low accumulating cadmium varieties of flax.
7.4.4.6 Seed Color

There is a growing market for yellow flaxseed in the baking industry and health
food trade. The market is expected to increase significantly because of the
interest shown in flaxseed as a dietary source of linolenic acid in human nutri-
tion. Most of the present market is in Western Europe with an increasing
market in Canada, Japan and USA. Both whole seed and flour are used in the
baking industry and yellow seed is preferred because of its aesthetic appeal in
bakery products. At the present time, there are varieties available in North
America that are grown under contract which are poorly adapted, disease
susceptible and low yielding (65–85% of brown seeded registered varieties).
Because of low yields a higher price is paid. Higher yields from improved
varieties will reduce the price and make Canadian flaxseed more competitive
in the world market and should result in higher production.
7.4.4.7 Disease Resistance

Flax diseases are a potential constraint to flax production in nearly all areas
where flax production occurs throughout the world. A significant portion of
flax improvement programs has been to increase resistance to pathogens and
thus to stabilize production and crop quality. The development of genetic
resistance is the most efficient and cost effective method to prevent losses in
yield and quality to diseases. Diseases that have received most attention are flax
rust, fusarium wilt, powdery mildew and pasmo and are caused by fungi.
Resistance to these diseases is under genetic control, and satisfactory sources
of resistant germplasm are available for use in flax breeding programs for flax
rust, fusarium wilt and powdery mildew. At this point in time a comparable
level of resistance to pasmo is not available. Breeding for resistance to some of
the diseases, especially the rusts in the past, has been hampered by continual
development and spread of new physiological biotypes of the causal disease
organism. Various sources of resistance to these diseases are available in flax
7 Flax 247
and are being introgressed into elite germplasm. Resistant germplasm, if prop-
erly used in conjunction with innovative breeding procedures, should provide
excellent disease protection in the years ahead. The development of molecular
markers for known resistance to flax pathogens will be extremely useful to
breeders and pathologists attempting to incorporate resistance to multiple
pathogens into high yielding varieties. The severity of crop loss to disease varies
significantly among regions and years because development and spread of
pathogens depends on various environmental conditions, the proportion of
the flax acreage planted to resistant cultivars, and the effectiveness of the
resistance.
7.5 Germplasm Sources
A wealth of diverse flax germplasm is available to flax breeders. The total

number of flax germplasm accessions existing in the world gene banks is
about 53,000 as shown in (Table 7.6). There are at least 81 genebanks, institu-
tions and breeding stations in the world, which maintain collections of flax
germplasm. The total number of flax germplasm accessions is large, but caution
should be used as there is a high degree of duplication between collections such
as those at the All Russian Flax Research Institute and the N.I. Vavilov
Institute for Plant Industry. In some cases germplasm accessions have been
integrated into breeding programs such as that of the German breeding com-
pany Deutsche Saatveredelung. Ethiopia has a large collection but access has
been restricted. The collections for flax germplasm of the United States
National Plant Germplasm System and Plant Gene Resources of Canada and
that of the Research Institute for Technical Cultures are to a great extent
Table 7.6 Genebank collections of Linum germplasm

Number of
Name of institution Country accessions
All Russian Flax Research Institute, VNIIL, Torzhok Russia 6,100
N. I. Vavilov Institute of Plant Industry, VIR, St. Russia 5,700
Petersburg
Research Institute for Technical Cultures, RITC China 4,000
Breeding Company DSV, Lippstadt Germany 3,500
Ethiopian Genebank, Addis-Ababa Ethiopia 3,100
North Central Plant Introduction Center, Ames, Iowa USA 2,800
Plant Gene Resources of Canada, PGRC, Saskatoon Canada 2,800
Research Institute for Cereals and Industrial Crops, Romania 2,700
RICIC, Funduela
Other collections (81) 22,300
Total 53,000
Source: Diederichsen and Richards (2003).
248 S.D. Duguid
duplicates of one another. According to Diedrichsen and Richards (2003) about

30% of the world flax germplasm can be considered unique. They suggest this
duplication helps to ensure preservation of diversity as continued preservation
in genebanks can be severely affected by political change, genebank priorities
and problems with rejunvenation. The wild species in the genus Linum are rarely
found in collections because they are perennials and are difficult to regenerate
due to cross pollination. The German genebank at Gatersleben has the largest
collection of these species.
Released cultivars are also maintained by the originating breeding program
or by the Plant Gene Resource Centre of Agriculture and Agri-Food Canada in
Saskatoon, Saskatchewan or by the United States Department of Agriculture.
Germplasm developed in the individual breeding programs is often exchanged
informally with other breeders or is formally released and registered to make it
available to all breeders.
7.6 Breeding Procedures
Flax breeding procedures usually have been grouped into three general
categories: introduction, selection and hybridization followed by selection.
Selection from introduced accessions and pedigree selection following hybri-
dization have been the predominant methods of flax breeding. Prior to 1936,
most of the cultivars recommended for production in North America were
derived by mass or single plant selection from introduced accessions.
Although many different breeding procedures and combinations of proce-
dures are used by flax breeders today, a universal feature of all current
programs is hybridization followed by selection. However, there are notable
exceptions: (1) Norland which was selected from Victory, a variety that was
heterogenous for both plant height and maturity; (2) Redwood 65 which
was selected from an x-irradiated population of Redwood and represented
an improvement in oil content; (3) Andro; (4) CDC Normandy somaclonal
variation from tissue culture and (5) CDC Triffid, a genetically transformed
cultivar with sulfonylurea herbicide resistance.
7.6.1 Selection of Parents
The initial step of any flax breeding program is to clearly define the
objectives which are both economically and biologically reasonable. Then
the breeder must choose the parents for creating segregating populations
from which a potential cultivar will be selected and to a great extent this
will be based on the specific breeding objectives of the program and avail-
ability of germplasm to meet those objectives. A flax breeder can only use a
small part of the parental material available; the success of the program
7 Flax 249
depends upon the ability of the breeder to select parents that will produce
superior progenies. The parents are then crossed. Flax breeders commonly
choose and hybridize the parents with complementary traits with the objec-
tive of selecting individual progeny lines that combine those traits.
7.6.2 Methods of Combining Parents

The methods used to make a cross and proper cultural practices have been
summarized by Beard and Comstock (1980). An unopened bud (prior to
anthesis) that has petals protruding 30–60 mm past the sepals is chosen as the
female parent and emasculated. Pollen from the male parent is transferred as
soon as the anthers dehisce. Typically, 70% of the pollinations result in viable
seed which are usually grown in the greenhouse or in the field.
An understanding of the genetics of desirable new traits is of practical value to
the flax breeder since such information greatly assists in determining breeding
objectives, developing breeding strategies, and utilizing resources efficiently.
Knowledge of the genetic relationships among different traits can also be useful
since desirable new traits may have undesirable pleiotropic effects on other impor-
tant traits or be linked to undesirable traits. New sources of resistance or other
desirable traits often occur in unadapted, agronomically inferior germplasm with
poor quality, and must be incorporated into a more useful genetic background
before they can be used in breeding programs. Rapid advances are currently being
made using molecular markers and successful transformations have been reported
in flax. These powerful tools have great potential to improve understanding of flax
genetics and accelerate the development of improved germplasm and cultivars with
novel genes. It is important to participate in these new developments if the flax
industry is to be competitive at home and abroad. However, much work still needs
to be done in identifying desirable new traits and determining their inheritance so
that biotechnological techniques can be effective.
The incorporation of desirable new traits into acceptable flax cultivars
requires considerable effort over a long period of time. The backcross method
is effective when the new trait is simply inherited, easy to detect and occurs in an
unadapted genotype. When unadapted germplasm is introgressed into adapted
germplasm for population improvement, one backcross to the adapted parent
for population improvement or a three-way cross are more effective than a
single cross. When both parents contribute to a number of desirable traits, a
single cross should be used with standard breeding methods, such as the
pedigree or bulk methods. Double crosses can be used to accumulate both
major and minor genes. Several generations of selfing and selection may be
required particularly if a number of minor genes are involved. A number of
breeding cycles may also be needed to introgress desirable traits such as lino-
lenic acid into an acceptable flax cultivar. A double haploid recurrent selection
procedure that takes only 2 years for each complete cycle has been devised.
250 S.D. Duguid
7.6.3 Methods of Breeding
The choice of breeding method to handle the segregating populations depends

upon factors such as the breeding objectives, nature of the crop, parental lines
available, number of traits, relationships among the traits, mode of inheritance
and ease of selection for a particular trait, diversity and number of parents and
resources available to the breeder. Methods commonly used in flax breeding
include the pedigree method with various modifications, bulk selection, back-
crossing, and single seed descent. While conventional breeding practices are
primarily used, anther culture or microspore techniques are also now being used
to accelerate the development of new flax cultivars and improved germplasm,
and to facilitate genetic studies.
7.6.3.1 Pedigree
The pedigree method is the most widely used flax breeding procedure. The
pedigree method is effective when the traits of interest in the breeding
program are easy to identify, highly heritable, and thus can be effectively
selected in early generations. For example, the pedigree system has been
especially effective in flax for rust resistance, oil content, and fatty acid
composition. The pedigree method of breeding, like any other method, has
certain advantages and disadvantages. The method is labour intensive and
more expensive than other methods, but it can systematized with the
assistance of computer programs to simplify the record keeping and data
collection so that large numbers of crosses can be handled efficiently. A
major advantage of the method is that undesirable progenies are elimi-
nated early, and only the superior progenies are advanced to the next
generation. The intensive selection in early generation selection may result
in undesirable limitations on recombination, especially in cases where traits
of interest are linked with undesirable genes for other important traits.
When using the pedigree method, the breeder emphasizes intensive selection
in segregating generations and the establishment of homozygosity and homo-
geneity within lines before entering them into advanced yield trials. This
method consists of making a cross between two genotypes possessing the
characters to be combined in a new variety and increasing the F1 seed in the
greenhouse, growth room or field. The main purpose of the F1 generation is to
provide sufficient seed for the F2 population, therefore, any method which does
this is quite satisfactory. The size of the F2 population will depend on many
factors including breeding objectives, availability of seed, genetic differences
between the parents, and resources available for selecting and handling indivi-
dual plants. Selection begins in the F2 generation and progenies from the
selected F2 plants are grown and selected in each subsequent generation until
near homozygosity or at least until individual lines appear uniform for visual
characters. Selection may be practiced both between and within progeny rows.
7 Flax 251
Visual selection is conducted for desirable agronomic characteristics such as

early maturity, plant height, disease resistance, high number of bolls which has
been shown to be a good indicator of yield potential. A common practice is to
select several plants from the progeny rows or hills that appear most promising
for the traits of interest and increase the seed in a winter nursery or greenhouse.
During the winter increase, the reserve seed is evaluated for resistance to rust,
oil content and fatty acid composition. Those plants which are rust resistant,
having high oil content and oil quality are grown to maturity in the winter
nursery/greenhouse. This cycle is repeated for each generation. The bolls from
the individual plant are threshed and maintained separately, examined visually,
and selected ones saved for the next generation. The same procedure is followed
until the desired level of homozygosity is reached. At this point, each selected
progeny row is bulk harvested and used for further seed increase and perfor-
mance testing. Most breeders do not increase individual lines for advanced yield
testing before the F5 to F7 generation.
The remaining lines are then evaluated in preliminary yield trials. Data
collected for days to maturity, plant height, 1,000 seed weight, lodging resis-
tance, oil content, oil quality and resistance to rust, powdery mildew and
pasmo. The lines are also grown in the wilt nursery to confirm resistance to
wilt. The preliminary yield trials serve to reduce the number of lines to a
quantity which can be handled in regional trials conducted by the individual
breeding institutions. These trials serve to determine the performance of experi-
mental lines in flax growing areas in Canada. The very best candidate lines from
each breeding institution are then entered into the Cooperative Test which is
grown at 20 locations across Canada with 15 locations in three Prairie Pro-
vinces. Each participating breeding institution, university or private industry
grows a uniform set of entries at each location and the data is compiled and
analyzed. These results are ultimately used in support of the licensing of
varieties in Canada.
7.6.3.2 Single Seed Descent

Single seed descent is a form of the pedigree selection method that is being used
by breeding programs to rapidly advance germplasm through segregating gen-
erations to produce varieties or generate genetic material for research purposes.
A single seed from each F2 plant and their progenies are advanced through
succeeding generations by harvesting one seed from each plant to provide the
population for the next generation. The procedure is repeated for several
generations to obtain a population of near-homozygous plants for selection.
Individual plants are then harvested and planted in individual rows and the
selected lines are harvested for further seed increase and testing. The single seed
descent method is especially well adapted for generation advance in the green-
house. No selection is generally practiced and since only one seed is harvested,
optimum plant growth is not required.
252 S.D. Duguid
7.6.3.3 Backcross
Generally, the backcross breeding method is used to transfer one or at most a
few genes controlling a simply inherited trait into an otherwise outstanding
cultivar or elite germplasm. The line possessing the desirable trait is the recur-
rent parent and the parent contributing the simply inherited trait is the non-
recurrent or donor parent. In a typical backcrossing program, the F1 of a single
cross is crossed back to the recurrent parent, and in subsequent backcrosses
only selected plants having the desired traits from the donor parent are crossed
back to the recurrent parent. With each successive backcross, the genetic con-
tribution from the donor parent is reduced by one half resulting in rapid
recovery of the recurrent parent. Most breeders believe that four to six back-
crosses are sufficient to recover the prototype of the recurrent parent. The
backcross method has been used to transfer single genes for disease resistance
from unadapted to adapted varieties of flax. This was typical of the approach
taken by flax breeders incorporating new rust resistance genes into varieties or
for the generation of genetic material such as the set of rust differentials
developed by Dr. H.H. Flor by backcrossing individual genes into the cultivar
‘‘Bison’’.
7.6.3.4 Bulk
In the bulk method, seed harvested from the F1 is bulk seeded in the field, and
the F2 is bulk harvested to provide seed for the next generation. The entire plot
from each cross is harvested in each generation, and a random sample of the
bulked seed is used to plant the next generation. This usually is repeated until F5
to F7 after which individual plants are selected from the bulk population. These
selections are grown in individual row or hill plots and lines with desired traits
are harvested and advanced to yield tests.
7.6.3.5 Doubled Haploids

Doubled haploids in flax can be produced by anther culture (Chen et al.
2003). All methods require the use of tissue culture techniques, precise
growing conditions, specialized technical skills, and considerable time and
effort. The efficiency of doubled haploid production will ultimately deter-
mine its success. There are large genotypic effects on response to anther
culture and the populations derived from anther culture may not represent
an unbiased sample of parental gametes which is a disadvantage in con-
ducting genetic studies. The use of doubled haploids would save the
breeding program 2–3 years in developing new cultivars and increase
cultivar uniformity. The complete homozygosity of doubled haploid lines
results in greater genetic uniformity of new cultivars and makes screening
for genes of interest more reliable. The capability to produce doubled
haploids would allow new cultivars to be developed more rapidly in
7 Flax 253
response to changing market demands, new niche markets and disease

problems. It provides ideal material to study the genetics of important
traits such as disease resistance and quality, and to develop molecular
markers to facilitate marker assisted selection when it becomes feasible.
While the use of doubled haploids has the potential to speed up and
increase the efficiency of plant breeding program, it is more costly to
implement, uses more growth facility space, and requires more highly
trained technical support than conventional plant breeding methods. As
a result, the breeder may not be able to select among as many lines per
cross in as many crosses, which may reduce the probability of success. It
may also be more difficult to break unfavorable linkages using doubled
haploids since there is less chance for recombination.
7.7 Summary
Canada leads the world in flax production and exports. Today, the unique
properties of flax differentiate it from other oilseeds and a new spectrum
of food, animal feed and industrial uses is expanding markets and adding
value to the crop. As demands of the industrial and functional food arenas
change, both nationally and internationally, further improvements in flax
production and bioproduct quality will need to occur in order to capture
the market. The flax breeding programs contribute to the increasing and
sustaining flax production by developing varieties and germplasm with
improvements in yield but also by reducing the risk of abiotic and biotic
stresses through the incorporation of genetic resistance. Further enhancing
the competitiveness of the cultivars and germplasm is the genetic improve-
ment of oil and meal quality along with the reduction in anti-nutritional
substances. This will create new opportunities for cultivars with specific
end uses which are environmentally friendly and safe.
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7 Flax 255
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Chapter 8
Cotton
Steve Hague, Lori Hinze, and James Frelichowski
8.1 Introduction
In the 19th century, uses emerged for cottonseed left over from ginning har-
vested cotton (Altschul et al. 1958). Oil from cottonseed was determined to be
useful in a range of food products, and cotton is now considered second only to
soybean in the value of its oil products. Seed of the cotton plant (Gossypium
spp.) generally has an oil content of around 21% and protein content near 23%.
The fatty acid profile of cottonseed includes about 55% polyunsaturated fatty
acids, 18% monounsaturated fatty acids, and 27% saturated fatty acids
(Lukonge et al. 2007; National Cottonseed Products Association 2007). Use
of cottonseed has been hampered because it contains gossypol, which is a toxic
compound. Nevertheless, ruminant animals can digest gossypol in limited
quantities, and it can be removed during oil crushing processes. Cottonseed
oil has good stability as cooking oil and can withstand high temperatures
without deterioration. Because of the stability of cottonseed oil, partial hydro-
genation is not necessary, which enables manufacturing of many non-transfat
products. Moreover, cottonseed is a very popular feedstock for ruminant
animals such as cattle. Cottonseed value as a protein source for dairy cattle
milk production is equivalent to that of other high quality oil seeds such as
canola (Brassica napus) and soybean (Glycine max) (Sanchez and Claypool
1983; Brito and Broderick 2007), and can be an economically lucrative alter-
native feedstock in areas where it is available (Blackwelder et al. 1998). While
the crude protein level of cottonseed is relatively high for a livestock feed, value
of the protein is limited by low levels of the amino acids methionine, threonine,
valine, and most critically lysine (Nagalakshmi et al. 2007).
Despite the extra profit from the sale of cottonseed and the emergence of new
markets for cottonseed, historical production practices have been focused
almost exclusively on fiber yield and quality because cottonseed is typically
S. Hague (*)
Department of Soil and Crop Sciences, Texas A&M University,
College Station, TX, USA
e-mail: shague@ag.tamu.edu

258 S. Hague et al.
only worth about 12% of the crop’s value (Elam 1995). There is an economic
disconnect between producers and incentives for higher quality seed. Producers
are paid for their cottonseed based on volume with little regard for quality
parameters. When prices for seed go up or down due to market value and/or
seed quality, economic consequences are spread across all producers who use a
common gin and not to a specific producer or even less so to a particular field of
cotton.
New market opportunities for cottonseed appear to be rapidly developing.
Cottonseed is a renewable fuel alternative to fossil fuels, and there is surging
interest in cottonseed oil for use as a biodiesel (Karaosmanoglu et al. 1999; He
and Bao 2005; Putun et al. 2006; Royon et al. 2006; Smith 2007). In addition,
new opportunities for food use are now possible because of recent biotechno-
logical advances in producing gossypol-free seed (Sunilkumar et al. 2006;
Townsend and Llewellyn 2007). The process currently used to remove gossypol
from cottonseed damages protein value (Freidman 1996). If seed can be pro-
duced without gossypol, then protein value of cottonseed should inevitably
improve.

Cotton is historically referred to four species of the genus Gossypium:
G. hirsutum, G. barbadense, G. arboreum and G. herbaceum, all of which
produce long fibers on seeds, suitable for spinning and weaving into fabrics.
These four species are cultivated between 378N and 328S latitude worldwide.
The genus Gossypium includes 44 recognized diploid (2n=26) and five
tetraploid (2n=52) (Fryxell 1992; Percival et al. 1999) species in drier
tropical and subtropical regions worldwide. Plants of this genus all produce
perfect, often highly visible flowers that are normally self-compatible
(Fig. 8.1). Cross-pollination is limited to insect vectors. Most species are
perennial shrubs or sub-shrubs adapted to flower and set seed according to
native photoperiod and rain seasons. This genus is distinguished from other
genera in the Malvaceae family by distribution of gossypol-producing glands
throughout plant tissues.
The genus is thought to have originated 10–20 million years ago when
continents were closer in proximity. Breakup of the continents divided Gossy-
pium into easily recognized groups of species and genomes that subsequently
evolved independently. In Australia, 18 species exist and are recognized by the
C, G and K genomes. In Asia and Africa, 13 species are found of the A, B, E and
F genomes. In Central and South America, 13 species of the D genome and five
species of the tetraploid AD genome are found. These genome types were
assigned by cytological observations of chromosome size, structure, and beha-
vior in meiosis with intra- and interspecific hybrids. Cytogenetic and molecular
evidence suggests that long ago a progenitor of each of the A and D genome
8 Cotton 259
Fig. 8.1 The cotton flower is a perfect flower having all male and female parts. Cross-
pollination generally is facilitated by bees, otherwise, the flowers will self-pollinate
species united to form a tetraploid AD genome (Wendel and Cronn 2003) that
subsequently evolved into the five tetraploid species known today.
Two A genome diploid species (G. arboreum and G. herbaceum) and 2
tetraploid species (G. hirsutum and G. barbadense) have been domesticated for
thousands of years in the Old and New World, respectively. Considerable
introgression and overlapping either within or between the new and old species
has occurred because of cultivation and manipulation by man. These four
species have become adapted to both feral and disturbed habitats and have
changed from perennial, often photoperiodic plants, to day-neutral annuals.
Popularity of these four species is attributed to seed fibers that are highly
elongated and to convoluted single cells that are mostly cellulose crystals at
maturity. Other species produce extremely poor fiber and, in some cases, no
fiber at all. The wealth of habitats and morphological diversity of undomesti-
cated species still provides ample genetic diversity for cotton taxonomic studies
and breeding.
8.2.1 Taxonomy
Early classification of cotton by Todaro (1877) and Watt (1907) applied many
taxonomic divisions to the morphological diversity in what is now known by
260 S. Hague et al.
just four species. Zaitzev (1928) was among the first to recognize separate
diploid and tetraploid species in the genus. Many subspecies were still applied
to these species and now are recognized as discrete lineages within each species
having distinct morphological and agronomic characters.
G. herbaceum has been described into five forms: acerifolium, africanum,
kuljianum, persicum, and wightianum (Hutchinson 1963; Brubaker et al. 1999).
Only var. africanum is recognized by taxonomists (Fryxell 1992) and considered
to truly exist in the wild. It is the most variable form and is found in southern
Africa. Acerifolium is the most primitive cultivated form that still grows as a
perennial in areas of Northern Africa and adjacent Arabia. The three other
forms are annuals in their environment: kuljianum in western China, persicum in
Iraq and the Levant, and wightianum in western penninsular India. G. arboreum
has been described into four mostly perennial forms (burmanicum, cernuum,
indicum and soudanense) and two annual forms (bengalense and sinense).
Burmanicum and cernuum originated in Northeastern India. Indicum is the
most primitive form and is found in western penninsular India and eastern
Africa. Soudanense grows to tree size in northwestern Africa and Sudan. Ben-
galense originates in Northern India and sinense originates in China, and both
are areas of extremely early maturity. China, India, and Pakistan still maintain
germplasm of these diploids that are valuable sources of trait improvement for
Upland cotton cultivated in arid environments.
G. barbadense originated in northwestern South America, but it has been
cultivated throughout South America and Central America. Originally,
G. barbadense was classified into three forms: barbadense, brasilense and darwi-
nii. The ‘moco’ or kidney cottons of Brazil were commonly associated with
brasilense but later were recognized as just a local cultivar of G. barbadense not
worthy of subspecies status. Darwinii was later classified separately as another
species of tetraploid cotton, G. darwinii. ‘Tanguis’ cotton varieties of Peru and
‘Sea Island’ types from the Caribbean and southeastern US are common early
types of G. barbadense. Muhammed Ali Pasha developed the foundation of
modern Egyptian cotton varieties by combining Sea Island varieties with other
strains of G. barbadense. The boll weevil (Anthomonous grandis, Boheman)
ended the G. barbadense industry in the southeast US because these cottons
were extremely susceptible to this pest. Despite a brief revival in that area,
G. barbadense cottons were soon relegated to Arizona and California. The
USDA started a breeding program in Arizona with G. barbadense and Egyptian
cottons to develop ‘Pima’ cottons. These became known as extra long staple
(ELS) cottons and now occupy a small, high quality cotton niche in the global
cotton market.
The first efforts to classify G. hirsutum led to a large array of taxonomic
subdivisions. As late as 1963, Hutchinson still recognized seven forms of
G. hirsutum: latifolium, marie-galante, morrilli, palmeri, punctatum, richmondii,
and yucatanense. Later taxonomists would disregard these subspecies names,
but their morphology and geographical distribution are still distinguishable by
these names and may hold clues to ancient cultivation and distribution by
8 Cotton 261
peoples in Central and South America. Marie-galante is strongly arborescent

and originated on the west coasts of southern Central America and northern
South America. It was likely distributed by natives and/or explorers to the West
Indies and northwestern South America. Punctatum describes smaller, bushier
G. hirsutum cotton found along the coasts of eastern Mexico, Florida, Yucatan,
and the Bahamas. It was discovered early by explorers and brought to West
African colonies where it naturalized and contributed to cotton breeding for
bacterial blight disease resistance. Morrilli describes G. hirsutum with bushy
growth, large and intensely hairy leaves that grows in the present day Mexican
States of Oaxaca, Puebla, and Morelos. The state of Guerrero contains
G. hirsutum form palmeri, with deeply dissected and glabrous (i.e., okra) leaves,
pyramidal growth, and strong anthocycanin production. In a restricted area of
northern Yucatan, G. hirsutum of the form yucatanense grows wild, whereas
most other forms usually grow in association with humans or in disturbed
areas. In the Mexican state of Chiapas and regions of Guatemala, G. hirsutum
form latifolium is found. These forms bear the most fruit in their first season,
and thus they are easily treated as annuals in cultivation. Most of the early
improvements in US cotton varieties depended on introductions of G. hirsutum
form latifolium.
8.2.2 Domestication
Archaeological evidence dates domestication at least as early as 2,000–3,000
BC for G. arboreum in regions of India (Gulati and Turner 1928) and for G.
barbadense in Peru (Stephens 1958). Each of the four cotton producing
species are thought to have been cultivated independently in their original
habitats and altered by human selection during cultivation and distribution
to new habitats (Hutchinson et al. 1947; Hutchinson 1951). The toxicity of
gossypol in seeds suggests that cotton was primarily grown for fiber rather
than food.
It is difficult to truly determine wild habitats or progenitors of these four
species due to extensive cultivation by man. For example, G. barbadense is
considered to have originated in northwest South America, but forms have
been found throughout South and Central America. In Brazil, local cotton
with the distinct characteristics of large leaves and kidney shaped seeds
appears to be a subspecies of G. barbadense, but it was selected for this growth
habit to facilitate easier harvest and removal of fibers. Conquests by Alex-
ander the Great helped reveal the existence of cotton fabrics in India to the
rest of the Old World. Continued development of trade routes to India
prompted cotton to be cultivated throughout the Old World and also
increased demand for products produced with cotton fibers. Great Britain
strongly influenced distribution and demand for cotton. The British estab-
lished the Empire Cotton Growing Company which sponsored cotton
262 S. Hague et al.
cultivation and improvement in areas they colonized (e.g. Sudan, Uganda,

and the West Indies). In Trinidad and Sudan, a large collection was estab-
lished of cotton cultivars and species collected by the Empire Cotton Growing
Company. Continued domestication of cotton adapted these four species to
regions outside their natural tropical habitats and even to more temperate
northern or southern latitudes.
European settlement of the Americas led to discovery and dispersal of
tetraploid species of cotton. Likely all four species were grown in the US, but
soon it became apparent the tetraploid species were superior in yield and quality
to the two Old World diploids (Watt 1907). In colonial North America, early
cotton culture was focused on limited cultivation for local uses. In mild coastal
climates of Georgia and the Carolinas, G. barbadense cultivars from the Car-
ibbean were cultivated and called Sea Island cotton. These cultivars were likely
derived from prior cultivation in the West Indies and produced very high
quality fiber but with limited yields. They were poorly adapted to other main-
land areas. Britain was among the few countries with a significant industry
manufacturing cotton products and needed to import cotton. British merchants
valued the high quality lint of G. barbadense. G. hirsutum was cultivated further
inland, throughout the Southeast and as far west as Texas, thus earning the
name ‘Upland’ cotton. ‘Georgia Green Seed’ and ‘Creole Black Seed’ were two
early varieties of Upland cotton planted in the South and Southeast. Creole
Black Seed was introduced from Siam into Louisiana by the French and was
easy to gin. Georgia Green Seed was introduced from the West Indies and had
better yields (Moore 1956). After the Revolutionary War, cotton was valued as
an export crop, but it was difficult to produce and process the quantities needed
for export. Invention of the Whitney Gin in 1793 was among many improve-
ments to make cotton production more profitable in the US. Development of
cotton in the US would have a large impact on the growth of the cotton industry
and cultivation worldwide.
In 1806, Walter Burling, a planter from Natchez, Mississippi, successfully
smuggled seed of valuable cotton germplasm out of Mexico City. This
germplasm and progeny resulting from its hybridization with Creole and
Green Seed was the first step to improving Upland cotton in the US. Better
quality fiber, earlier maturity, and resistance to diseases were noticed in the
newly created cultivars (Wailes 1854). Burling’s Mexican cotton was grown in
the Mexican Highlands and exhibited earliness and an annualized growth habit,
which distinguished it from most G. hirsutum. It likely represented significant
progress by Mexicans or their fore bearers to adapt G. hirsutum to cultivation.
Local reselection within this novel germplasm enabled adaptation and improve-
ment of Upland cotton in the US. Early cotton breeders practiced visual
selection for lint quality and quantity and eventually learned to select the best
individual plants (Moore 1956). Large, easily picked bolls with long fibers were
emphasized in selection of cottonseed for local cultivation. In time, four general
types of cotton came into use in the United States, and they were named
8 Cotton 263
according to their general geographical areas of production: ‘Eastern Big Boll,’

‘Delta,’ ‘High Plains’ and ‘Western Acala’ (Ware 1951; Smith et al. 1999).
Emergence of pests and diseases would shape further domestication of
cotton in the United States. Infestation by the boll weevil in the late 1800s
prompted a large scale change in cotton germplasm and practices. Left
unchecked, the boll weevil could easily render most US cotton fields completely
barren. Vital cooperation among federal, state, and private institutions helped
to combat the boll weevil and preserve the cotton industry. Early maturing
cotton crops were the best strategy for coping with the boll weevil at this time.
Lint yield and fiber quality were sacrificed in order to produce a harvestable
crop before boll weevil pestilence became too great to produce a profit. Due to
increased boll weevil pressure later in the growing season, public and private
cotton breeding programs shifted to developing early-maturing germplasm.
The federal government sponsored plant explorations into Mexico, the natural
habitat of G. hirsutum, in the hopes of finding host plant resistance to boll weevil
and potential for cotton breeding improvement. Substantial genetic resistance
to the boll weevil was never identified, but Burling’s original Mexican Stock and
other Mexican cottons were imported to the US to improve earliness, yield, and
harvestability of cotton varieties.
Reduction in cotton production from the Civil War and boll weevil out-
breaks gave impetus to other nations to increase cotton cultivation to meet
worldwide demand for cotton (May and Lege 1999). These countries without a
doubt benefited from Upland cotton varieties produced in the US and from
varieties and species distributed worldwide. Upland cotton production became
established in Africa, Southeast Asia, Egypt, South America, portions of the
Mediterranean and the Middle East. The former USSR had already explored
Central America for cottons to augment their cotton breeding programs
(Mauer 1930). India, China, Pakistan, and other countries were replacing Old
World and obsolete tetraploid varieties with Upland varieties developed in the
US and elsewhere. Many of these countries benefited from increased earliness in
Upland cottons in order to expand cotton production to temperate areas. With
an emerging global cotton market and improved ginning and spinning technol-
ogies, the US cotton production industry is now faced with the need to increase
the quality and productivity of its cotton.
8.3 Varietal Groups
G. hirsutum cultivars comprise over 90% of the cotton grown worldwide,

followed by G. barbadense, and to a very limited extent, G. arboreum and
G. herbaceum in remote localities of Asia and Africa. As stated earlier, Upland
cotton (G. hirsutum) grown in the US can be broadly divided into four groups:
Eastern Big Boll, Delta, High Plains, and Western Acala (Ware 1951; Smith
et al. 1999). Eastern Big Boll reflects an early emphasis on large bolls in the
264 S. Hague et al.
east-southeast part of the Cotton Belt. Delta cottons often were grown in the
fertile alluvial soils in the Mississippi River floodplain and sported high-yield-
ing, high-quality crops. Due to the long growing season and therefore large
plant sizes often obtained in this region, more emphasis could be placed on
yield. High Plains cottons are characterized by more compact growth habits
with stormproof bolls that retain cotton fibers to prevent losses due to wind or
rain and by adaptation to partial or no irrigation. Stripper harvest machinery
was engineered to remove cotton from the stormproof bolls. Western Acala
cottons were developed from high-quality cottons collected from the Acala
region of Chiapas, Mexico. Acala cottons are primarily grown in Arizona,
California, New Mexico, and western Texas. They are well-adapted to long,
dry summers and irrigation practices. Fiber quality of these cottons command
a premium compared to most other Upland cottons. California became noted
for mandating a one-variety law for Acala cottons in order to distinguish
themselves from other cotton producing states. This action secured a premium
for all California growers. Over time, historical distinction among cotton
variety groups became blurred with the advance of the boll weevil, which
made it necessary to grow early-maturing varieties in both the eastern and the
southern regions of the Cotton Belt. Pedigrees of these four groups became
more intertwined with additional needs for disease resistance and uniform
fiber quality. Bacterial blight, Fusarium and Verticillium wilts, nematodes,
etc. would affect large areas of the Cotton Belt and require simultaneous
improvement of much of the regional germplasm base. Also, formation of
national and global seed companies restricted some local genetic diversity but
also integrated novel gene sources (i.e. recombinant DNA technology) into
their cultivars (Stewart 1995).
Interspecific hybrids have demonstrated potential for combining the high
yield of G. hirstum and the quality of G. barbadense. Additional labor for
manual pollinations and/or development of a male sterility trait necessary to
facilitate production of hybrid seed has prevented widespread adoption of
commercial hybrid seed production in the US. However, in India and China,
cheaper labor and integration with value-added recombinant DNA traits
(transgenic technology) has allowed the prospects of hybrid cotton to remain
positive (Barwale et al. 2004; Dong et al. 2004).

Cotton germplasm of the four cultivated species were enhanced in their natural
ranges long ago by human cultivation (Hutchinson 1963). A mix of economic-
ally acceptable and wild forms still exists for three of these species in their
natural tropical ranges (Brubaker et al. 1999). Only cultivated forms of
G. arboreum have been found. The earliest known collection of cotton was
established by J.P.B. von Rohr in the late 18th century under a commission
8 Cotton 265
from the Danish King. He maintained a garden of cottons from the Caribbean
and South America at St. Croix in the Danish West Indies (now the Virgin
Islands) (Rohr 1791–1793). In the mid 19th century, the Italian botanists,
Parlatore and Todaro, assembled a collection of cotton (Todaro 1877). The
first major contributor to genetic resource accumulation and disbursement was
the former Empire Cotton Growing Company, chartered by Britain to grow
cotton in areas settled outside the US. This company introduced cotton to
Africa, the Middle East, and Australia, and established germplasm collections
in Trinidad and Sudan. Upon the closing of this company, the Trinidad collec-
tion was dispersed and the Sudan collection was maintained by the Sudanese
Department of Agriculture. A number of major cotton collections exist world-
wide that each contains thousands of accessions of cultivated and wild species
of cotton. Some of these major collections are located in the following coun-
tries: China, the former USSR (Vavilov Institute of Research, Tashkent and
Uzbekistan), India (Central Institute for Cotton Research in Nagpur), Pakistan
(Cotton Research Institutes of Multan and Sakrand District), France (Institute
de Recherches du Coton et des Textiles Exotiques), and the US. The US Cotton
Germplasm Collection is the largest single collection of cultivated and wild
cotton species worldwide (Percival et al. 1999). This collection contains over
9,300 accessions of 46 species of Gossypium from 101 countries or political
jurisdictions and from a range of latitudes from 408N to 408S. Even remote
tropical areas, such as the Galapagos Islands, harbor valuable germplasm and
continued exploration into these regions is justified. The collection historically
has been grouped into seven sub-collections:
1. Variety Collection: over 3,100 accessions of obsolete or publicly donated
G. hirsutum cultivars; originally established by the Delta Branch Experiment
Station in Stoneville, Mississippi; accessions are catalogued with the prefix
‘SA’;
2. Primitive Race Collection: over 2,100 accessions of wild or primitive races of
G. hirsutum; created at the Texas A&M University Experiment Station in
College Station, Texas, from plant explorations and germplasm exchanges
with other collections; accessions are prefixed by ‘TX’;
3. G. barbadense Collection: over 1,500 accessions of primitive germplasm,
obsolete and current cultivars; originally established at the Cotton Research
Center in Phoenix, Arizona; accessions are prefixed by ‘GB’;
4. Asiatic Collection: includes accessions of G. herbaceum (194 accessions, ‘A1’
prefix) and G. arboreum (1,729 accessions, ‘A2’ prefix); established at College
Station from germplasm exchanges;
5. Wild Species Collection: over 500 accessions representing 42 species of wild
diploid and tetraploid Gossypium species; established at College Station
from explorations, exchanges and donations; accessions are prefixed by the
genome designation of the species;
6. Genetic Marker Collection: includes G. hirsutum and G. barbadense lines of
special interest; and
266 S. Hague et al.
7. Base Collection: required backup collection that includes permanent storage

of all materials listed above and any new Plant Introductions; all materials
are maintained at the National Center for Genetic Resources Preservation
(NCGRP) in Fort Collins, CO.
Access to the US collection is freely available worldwide to persons with
a bona fide interest in cotton. In 2006 alone, seed of 4,160 accessions were
distributed to 105 researchers and institutions worldwide. Seeds are stored
at 48C and 23% relative humidity to prolong seed viability. Seed propaga-
tion routinely is performed on a subset of the collection to increase the
seed viability and quantity available for distribution. A longstanding
arrangement between the USDA, the National Cotton Council, and INI-
FAP (the Mexican agricultural ministry) established a cotton winter nur-
sery in Mexico for mass seed propagation of the germplasm collection.
This location is ideal for seed and fiber production, especially of photo-
periodic or slow maturing accessions. Other species are intensively cared
for in greenhouses to generate the necessary seed. Descriptor, fiber quality,
and agronomic evaluation data on accessions in the collection is shared
with researchers to assist in reviewing and requesting seed of cotton
germplasm.
In 1946, ‘A Memorandum of Understanding’ was formalized by 11 State
Agricultural Experiment Stations (SAESs) and the USDA (Cotton
Division). This established yearly meetings to arrange research priorities in
cotton. One major outcome was the development of germplasm resources
for cotton. The station at Stoneville, MS, was assigned the task of accumu-
lating obsolete cultivars of G. hirsutum, while the station in College Station,
TX, was assigned to maintain G. hirsutum and other species collected from
the wild. The station at Phoenix, AZ, focused on G. barbadense accessions.
Beginning in 1960, collections along with their characterization data were
deposited at NCGRP (formerly the National Seed Storage Laboratory). The
International Board for Plant Genetic Resources established a set of cotton
descriptors in 1980. All these collections were combined in 1985 at the
USDA-ARS facility in College Station, TX. This collection focused
worldwide with emphasis on germplasm exchange and conservation, while
still fulfilling the mission of maintaining the germplasm for use in cotton
improvement.
This germplasm resource is utilized extensively with worldwide requests
for germplasm and numerous trait evaluations and introgressions into
improved cotton germplasm. Tri-species hybrid crosses were made to intro-
duce increased fiber strength into Upland cotton (Fryxell 1976). Resistance
to bacterial blight (Brinkerhoff 1970), nematodes (Robinson and Percival
1997), pyramiding of multiadversity resistance (El-Zik and Thaxton 1989),
development of male sterility for hybrid production (Meyer 1975) and
evolutionary studies (Wendel and Cronn 2003) are just a few of the
accomplishments made possible with this germplasm resource.
8 Cotton 267
Whether improving yield, fiber quality, or reducing the cost of production,

increasing profit per acre has been the ultimate objective for cotton breeders. In
1900, average cotton fiber yield in the US was 195 lbs. per acre (Ware 1951); by
2005, it was 831 lbs. per acre (Meyer et al. 2006). While not all the gain can be
attributed to genetic improvement, certainly cotton breeding programs deserve
much credit. Meredith and Wells (1989) reported that in comparing modern
cultivars to antiquated cultivars, newer germplasm is capable of higher yields
because of greater partitioning of dry matter to reproductive structures and
away from vegetation. Certainly, specific improvements in cotton cultivar yield
beyond a more favorable ratio of fruiting to vegetative tissue have led to this
dramatic improvement. Areas of improvement include host plant resistance,
abiotic stress tolerance, agronomic adaptation, improved fiber quality, and
enhanced seed traits.
8.5.1 Host Plant Resistance

Several insect, nematode, and disease maladies afflict cotton. Perhaps none is as
damaging as the boll weevil. Its invasion of the US Cotton Belt at the end of the
19th century financially ruined much of the cotton industry. The boll weevil
created a societal upheaval and prompted scientists to begin searching for host
plant resistance to this pest (Jones 2006). While no cotton germplasm provided
substantial genetic resistance, earlier maturing varieties were developed that
provided some temporal resistance to the boll weevils (Walker 1979). By devel-
oping cotton varieties that produced a greater portion of their fruit before the
2nd and 3rd generations of post-diapause boll weevils could reproduce, an
economically acceptable cotton cultivar could be grown in many areas of the
US Cotton Belt.
In the 1960s, efforts to identify mechanisms of resistance to other insect pests
were sought. Several biochemical and morphological traits associated with
cotton host plant resistance were identified with varying degrees of usefulness
(El-Zik and Thaxton 1989). Butler and Henneberry (1984) determined that
fewer leaf trichomes resulted in greater cotton host plant resistance to the
whitefly (Bemisia tabaci). Increased tannins (Lege et al. 1992) and gossypol
(Bottger and Patana 1966), as well as glabrousness (Lukefahr et al. 1971) and
the nectariless trait (Butler et al. 1972) all reduced Heliothis spp. damage.
Schuster et al. (1976) reported that plants without floral nectaries also were
found to be less attractive to plant bugs (Lygus hesperus). The okra-leaf trait
confers resistance to pink bollworms (Pectinophora gossypiella) (Wilson et al.
1986), and it is a valuable means of improving pesticide penetration into the
lower cotton canopy (Fig. 8.2).
268 S. Hague et al.
Fig. 8.2 Upland cotton leaf shapes include the normal shape on the left, the okra-leaf shape is
on the right, and in the middle is the heterozygous intermediate-shape
Considerable breeding efforts have been directed towards improving

nematode and disease resistance of cotton germplasm. R.L. Shepherd (1979)
outlined methodology for screening germplasm for resistance to root-knot
nematodes (Meloidogyne incognita) as well as developing several resistant
cotton lines. Improving resistance to Verticillium wilt has been a major goal
of several breeding programs throughout the world. In areas where Verticil-
lium is prevalent, breeders often establish highly inoculated nurseries to screen
early-generation lines. Occasionally, breeders are even capable of developing
host plant resistance to poorly defined diseases. In 1995, a new and devastat-
ing disease, Bronze wilt, afflicted hundreds of thousands of US acres (Bell
et al. 2002). Although Koch’s postulates were never satisfied, most breeders
realized a strong relationship between cotton varieties and disease incidence.
Consequently, breeders soon purged much of their breeding material that was
susceptible to Bronze wilt. No major outbreak of the disease has been reported
since 2000. Breeders have successfully used host plant resistance against other
cotton diseases including bacterial blight (Xanthomonas campestris), Fusar-
ium wilt, reniform nematodes (Rotylenchulus reniformis), blue disease, and
boll rot.
8.5.2 Abiotic Stress Tolerance

Soil moisture deficits generally are the most yield-limiting stress factor for
cotton. In certain growing areas, other abiotic stresses are of major
8 Cotton 269
concern,including excessive salt and extremely high or low temperatures.

Abiotic stress screening usually entails subjecting early-generation lines to
extreme drought, heat, or cold conditions; and then selecting the most
productive plants to advance in breeding programs. Often, all advanced
lines in a program are tested under drought conditions to evaluate
performance. Heat tolerance generally is identified in plants by rating
pollen production. Breeding programs approach screening for heat toler-
ance at different stages of cultivar development. Some emphasize screen-
ing early-generation lines that are still segregating, while others wait to
identify tolerance or sensitivity until later generations. Seed increases are
grown in extreme high-temperature areas of Arizona to help identify
tolerance or sensitivity. Cool temperature stress typically receives little
attention during early stages of development. High levels of cold toler-
ance are often associated with larger seed size and tolerance to seedling
disease complexes. For salt tolerance, usually minor efforts are made for
selection and improvement. Gossett et al. (1994) indicated varietal differ-
ences between New Mexico State University’s Acala varieties and those
varieties developed in the Mid-South, which suggests serendipitous
enhancements for salt tolerance due to the New Mexico breeding pro-
gram environment.
8.5.3 Agronomic Adaptation
Early maturity can enhance yield and reduce the risk of late-season pest
damage and aberrant weather in areas with a short growing season. Con-
versely, late-maturing varieties can improve drought tolerance (Dumka et al.
2004). In latitudes closer to the equator, later-maturing varieties also can
exploit more heat units from the growing season, recover from fruit shed-
ding, and translate that opportunity into greater yield potential than more
determinant fruiting varieties.
The ideal boll type depends on location. On the High Plains of Texas and
Oklahoma where strong windstorms frequently occur, a tight storm-proof
boll is preferred. Necessity for such a boll, however, has decreased with the
adoption of chemical harvest aids, which shorten exposure time between
defoliation and harvest. In the Mid-South and Southeast, growers prefer
‘looser’ bolls, which are less prone to hard-locking and will ‘fluff-out.’ This
type of boll can be easily pulled out of the burr by mechanical pickers,
which results in greater harvesting efficiency and higher quality fiber with
less trash. In addition, bolls need to open easily in the presence of low
sunlight and high humidity, which are common in these regions during the
harvest season (Fig. 8.3).
270 S. Hague et al.
Fig. 8.3 A fully mature cotton plant has open bolls that are ‘fluffed-out’ and ready to
harvest
8.5.4 Fiber Quality
Because cotton’s ultimate value is as a fiber crop, much attention has been given
to improving fiber traits. Fiber quality is more heritable than yield potential and
can affect crop quality in the most environmentally diverse and challenging
conditions (Krieg 2002). Fiber quality has dramatically improved in almost all
US production regions. By the early 1960s, high-volume instrument (HVI)
testing was introduced and changed the way cotton fiber quality was measured.
With this advent, breeders could affordably evaluate fiber from individual
plants and breeding lines, and then assign absolute and objective numbers to
fiber properties. This technological innovation helped make rapid improve-
ments in fiber traits.
Concomitant with HVI development, new strategies were developed to
break negative linkages among fiber properties and yield (Miller and Rawl-
ings 1967; Meredith and Bridge 1971; Culp and Harrell 1973). Fiber length
and strength typically were characteristics most often targeted for improve-
ment because of their importance to spinning quality, quantification by
HVI, and the existence of alleles for fiber quality improvement. Today,
many breeders employ sophisticated crossing schemes designed to break
linkages between yield and fiber qualities. Other fiber properties such as
8 Cotton 271
micronaire, elongation, length uniformity, fineness, and maturity also garner

attention from breeders. Because of the low cost and accuracy of fiber analysis,
even on a single plant basis, most lines are evaluated during every generation of
development.
8.5.5 Seed Traits
Minor breeding attention has been given to improving seed quality because seed
represents only a small fraction of a cotton crop’s total value. Seed size typically
has been a trait receiving the most attention by breeders because small seed size
can result in poor seedling vigor (Quisenberry and Gipson 1974). More impor-
tantly, small seeds can cause problems at the gin by increasing seed coat
fragments and neps (Bargeron and Gardner 1991). Cotton breeders monitor
seed size through a seed index, which is the weight of 100-fuzzy seeds. Smaller
seed size is often associated with a greater lint-to-seed ratio, which is an
important yield component. Therefore, a tendency exists among breeders to
opt for smaller seed size as a means to improve yield.
A major devaluating characteristic of cottonseed is gossypol, a toxic second-
ary metabolite. Gossypol makes oil extraction and utilization more expensive
and less competitive with other oil crops such as soybeans and canola. The first
cotton variety with glandless seed was described by McMichael (1959). Less
than two decades later, several commercial glandless varieties were available
(Halloin et al. 1978), but none of these lines was very successful because of pest
damage and lack of value returned to growers.
Breeding goals vary depending on organizations and their missions. Cotton

breeding programs typically fall into three categories: private, state or provin-
cial, and federal. In the United States, private seed companies focus on short-
term, high-value objectives. State university programs address regional pro-
blems with development of germplasm and varieties. Federal US Department
of Agriculture (USDA) breeding programs conduct long-term improvement
programs and more basic research projects that have a broad, national scope.
8.6.1 USDA
Early on, the national interests of cotton were promoted by the USDA with
creation of the Division of Cotton and other Fiber Crops and Diseases. USDA
began sponsoring germplasm exploration trips in the early 1900s to the centers
of cotton diversity. These efforts resulted in nearly 1,000 accessions added to the
272 S. Hague et al.
national collection (Percival and Kohel 1990). Later, USDA breeding stations
were established to address the need for cotton varieties with greater yield and
better fiber qualities in Shafter, CA; Las Cruces, NM; Florence, SC; and
Sacaton, AZ. Once fiber quality was improved; however, maintaining the
high quality properties of these cottons after ginning became a problem. In
response, USDA created uniform testing procedures to class cotton fiber as well
as classing offices to assist plant breeders in predicting spinning performances
of breeding lines.
In 1943, the USDA-Agricultural Research Service (USDA-ARS) became the
dominant agency to conduct crop research within the USDA organization.
USDA-ARS plays a major role in cotton improvement along with plant intro-
duction, exploration, and germplasm maintenance activities. Through multi-
state research activities, the USDA-ARS, universities, and other stakeholders
coordinate activities to evaluate and develop cotton germplasm and genetic
resources the industry can use to develop cultivars. Some of the accomplish-
ments and ongoing efforts include:
– Release of over 100 breeding lines, often in concert with universities, with
enhanced fiber properties (CCGC 2005).
– Development of cytogenetic stocks useful as molecular breeding tools and
sources of diverse and valuable alleles.
– Creation of a saturated genetic map whereby genes associated with traits
can be identified in the genome and tagged for possible selection
(Frelichowski et al. 2006; Ulloa et al. 2007).
– Release of lines converted from photoperiodic landraces into day neutral
forms.
– Introgression of Upland cotton and G. barbadense to produce substitution
lines (Saha et al. 2006), aneuploid stocks for genetic mapping, and
improving yield and quality of both species (Percy et al. 2006).
– Evaluation and breeding for resistance to nematodes and Fusarium wilt.
– Continued expeditions into Mexico to explore for additional Gossypium
species.
– Negotiations with Chinese and Uzbekistan authorities for access to at
least 1,000 accessions from their respective cotton collections.
– Evaluation of cultivated and exotic breeding lines for resistances to insects
and heat stresses.
– Investigations of variability of cottonseed properties and sources of
improvement (Stipanovic et al. 2005).
8.6.2 State Universities

The establishment of land grant universities subsequent to the Morrill Acts of
1862 and 1890 began the involvement of universities in cotton research. State
universities disseminate information about agricultural advances to producers,
8 Cotton 273
apply the scientific method and innovation to solve problems related to cotton,
and educate and train the future labor force to support the cotton industry.
State Agricultural Experiment Stations (SAES) are branches of US university
systems. Phenotypic cotton breeding programs of SAES exist in New Mexico,
Texas, Louisiana, Arkansas, Mississippi, Georgia, and North Carolina. Private
cotton breeders conduct research into traits they deem to be the most profitable
for the companies they represent (i.e. yield, fiber quality, transgenic herbicide
and insect resistance). Public breeders generally cannot compete in this arena
because of the resources available to private breeders; therefore, public breeders
embrace research into characteristics with less industry-wide, but more regional
importance, while developing breeding lines and cultivars with acceptable
values for yield and fiber quality measures. Reviews of cotton breeding research
activities and objectives were documented by Bowman (1999) and Calhoun and
Bowman (1999).
Some breeding programs concentrate on identifying useful traits in obsolete
and wild cottons accessed from germplasm collections. Traits being evaluated
in breeding programs include insect and disease resistance and tolerance to
environmental stresses including drought, salt, and cold temperatures (Basal et
al. 2005). Such traits could be novel sources of variation for breeders to use in
developing new cultivars. Other programs focus on enhancing germplasm to
improve yield and fiber characteristics (i.e. improved fiber length and strength,
reduced micronaire and short fiber content, etc.). Some breeding programs
release cultivars while others release unique germplasm useful to other public
and private breeders. One of the most understated roles of the SAES breeding
programs is training students to become plant breeders.
State and federal institutions play a vital role in developing technologies and
germplasm resources that have been adopted by private companies. These
companies have assumed an increasingly larger role in cotton breeding world-
wide through the further enhancement of these resources to generate savings,
profits, or greater utility for the cotton industry (Heisey et al. 2001).
8.6.3 Private Companies
Since most cotton acreage in the lucrative markets of the United States,
Australia, Brazil, and Argentina are planted to transgenic cotton cultivars,
the primary focus of commercial seed companies is to provide a mechanism
of delivering technology to cotton growers and capturing value from seed
technology as revenue from chemical technology decreases. Most transgenic
cotton in the United States has a herbicide-resistant transgene and a transgene
for producing the proteins of Bacillus thuringiensis (Bt) (Table 8.1; USDA 2006).
Current commercial cotton planting seed markets have injected tremendous
profits for seed companies capable of delivering transgenic technology. More
than ever, speed of delivering new and improved cotton cultivars with transgenic
Table 8.1 Upland cotton acreage planted to genetically engineered (GE) varieties planted by state and across the United States, 2000–2007 (USDA-
274
NASS estimates)
Insect-resistant (Bt) only Herbicide-tolerant only
State 2000 2001 2002 2003 2004 2005 2006 2007 2000 2001 2002 2003 2004 2005 2006 2007
Percent of all upland cotton planted
Alabamay 10 10 10 28 25 25
Arkansas 33 21 27 24 34 42 28 32 23 29 37 25 15 12 21 16
California 3 11 6 9 6 8 9 4 17 27 26 27 39 40 40 51
Georgia 18 13 8 14 13 29 19 17 32 43 55 32 23 11 13 10
Louisiana 37 30 27 30 26 21 13 17 13 14 9 15 7 10 13 11
Mississippi 29 10 19 15 16 14 7 16 13 15 22 16 23 23 22 19
Missouriy 20 32 13 59 40 63
North Carolina 11 9 14 16 18 17 19 13 29 37 27 29 27 24 19 16
Tennesseey 13 16 10 8 10 17
Texas 7 8 7 8 10 14 18 16 33 35 40 39 40 35 34 36
Other Statesz 17 18 19 18 22 18 21 27 21 33 35 32 24 26 24 20
US 15 13 13 14 16 18 18 17 26 32 36 32 30 27 26 28
Stacked gene varieties All GE varieties
Alabamay 54 60 60 92 95 95
Arkansas 14 28 26 46 45 42 45 47 70 78 90 95 94 96 94 95
California 4 2 1 3 7 5 8 6 24 40 33 39 52 53 57 61
Georgia 32 29 30 47 58 55 64 68 82 85 93 93 94 95 96 95
Louisiana 30 47 49 46 60 64 68 68 80 91 85 91 93 95 94 96
Mississippi 36 61 47 61 58 59 69 62 78 86 88 92 97 96 98 97
Missouriy 16 25 23 95 97 99
North Carolina 36 38 45 48 46 54 60 64 76 84 86 93 91 95 98 93
Tennesseey 75 67 71 96 93 98
Texas 6 6 4 6 8 14 18 28 46 49 51 53 58 63 70 80
Other Statesz 36 33 32 38 45 46 45 42 74 84 86 88 91 88 90 89
US 20 24 22 27 30 34 39 42 61 69 71 73 76 79 83 87
S. Hague et al.
y
Estimates published individually beginning in 2005.
z
Includes all other states growing Upland cotton.
8 Cotton 275
technology is imperative to the profitability of private seed companies. Conse-

quently, great resources are made available to commercial breeding programs to
hasten development of transgenic cultivars.
The next generation of commercial transgenes will likely be for additional
insect pest control, abiotic stress tolerance, and unique fiber properties. Aside
from transgenic traits, commercial breeding objectives are improving lint yield,
fiber properties, and agronomic adaptability. Little regard is given to seed
qualities. Private companies strive to develop cultivars that are adapted to the
broadest possible range in an effort to limit their product portfolio and simplify
seed production operations.

Breeding methodology is standard among programs whether they are public
or private. The greatest differences between public and private programs
generally are the greater size and scope of private programs and their use of
transgenic technologies. Nevertheless, basic techniques of creating genetic
variability, selection practices, and evaluation procedures are similar. Cotton
is an autogamous crop, and breeding methods used in cotton are variations
on or combinations of methods normally utilized with self-pollinating spe-
cies (Fehr 1991).
Early generations of breeders employed mass selection within a heteroge-
neous population to improve cotton. With this technique, the breeder selected
individual plants of the desired phenotype, and sowed seeds of these plants for
the next generation. Then the breeder again selected individual plants with the
desired phenotype. This selection cycle was repeated to increase the percentage
of desirable genotypes while forming a homozygous population.
Most cotton breeders today use a pedigree method of breeding, first
described by Newman (1912), or slight derivations thereof. After initial
hybridization, counter-season nurseries, especially for first filial increases, are
frequently used to hasten development. Individual plants are selected in second
filial or later generations after additive genetic effects are expressed. Initial
testing of selected lines is done in well-controlled, high-yield potential environ-
ments where the greatest expression of alleles contributing to yield and fiber
traits is possible. As testing continues on advanced generation lines, trials tend
to include more diverse growing environments to determine performance
stability. The entire process generally takes at least ten years from an initial
cross-pollination to a finished cotton cultivar.
A yield plateau was thought to have been reached in the late 1990s due to
several factors. Chief among these factors was a lack of genetic diversity caused
in large part to, as Meredith (2002) suggested, breeding efforts directed toward
added value traits. Genetic diversity is an important component for advancing
breeding populations of cotton germplasm. Commercial plant breeders are
276 S. Hague et al.
pressed to quickly release high-performing varieties. Consequently, private

breeders tend to use more in-house elite germplasm with a narrow genetic
base in comparison to public cotton breeders (Bowman 2000). Commercial
cotton breeding programs are becoming more global in nature. Major cotton-
seed companies such as those owned by Monsanto, Bayer, and Dow recently
established cotton breeding stations around the world. These operations are
now exchanging breeding lines, which will enhance diversity and increase
opportunities for quantitative genetic improvement. In addition, their extensive
testing networks allow great insight into performance capabilities of elite
germplasm.
The cost of commercial cotton breeding has risen dramatically in recent
years due to transgenic technology. Concomitantly, return on investment has
risen as well. In some cases, the amount a US cotton grower pays for transgenic
cottonseed is 2000% more than they were paying for conventional cottonseed in
the early 1990s. This has had profound effects on the cottonseed industry. Most
seed companies are now owned by large chemical companies willing to invest in
biotechnology to offset losses from chemical sales. Because almost 90% of
cottonseed sold in the United States contains transgenic technology (USDA
2006) and much of the rest of the world is rapidly adopting transgenic cotton
planting seed (Table 8.2); commercial seed companies focus heavily on integrat-
ing the latest transgenic traits into elite lines.
The most common breeding procedure used to develop transgenic cultivars is
the backcross method. This technique involves mating a donor parent with the
transgenic trait of interest to a recurrent parent with the most agronomic traits
of interest. Breeders are provided donor plants with the transgene of interest.
To date, all commercial transgenes for cotton are independently segregating,
dominantly inherited genes. Breeders are also given tools to identify the
Table 8.2 Global acres (millions of hectares) of transgenic cotton (Bt and Bt/Herbicide
Tolerance) grown from 1996 to 2006
Year Bt Bt and herbicide Total
1996 0.8 0.0 0.8
1997 1.1 <0.1 1.1
1998 1.4 0.1 1.5
1999 1.3 0.8 2.1
2000 1.5 1.7 3.2
2001 1.9 2.4 4.3
2002 2.4 2.2 4.6
2003 3.1 2.6 5.7
2004 4.5 3.0 7.5
2005 4.9 3.6 8.5
2006 8.0 4.1 12.1
Source: International Service for the Acquisition of Agri-Biotech Applications (ISAAA
2006). http://www.isaaa.org/RESOURCES/PUBLICATIONS/BRIEFS/35/pptslides/
default.html
8 Cotton 277
transgene event in the way of Southern Blot assays, polymerase chain reaction
(PCR) technology, and/or lateral flow strips. This enables breeders to identify
the transgene throughout the breeding process. With PCR technology, identi-
fication of homozygous plants is possible, too. Generally two to three back-
crosses are made to a non-transgenic line with superior production potential.
Sometimes transgenic sister-lines are performance tested, and the best lines
are bulked together to form the new transgenic cultivar. Other times, all
available backcrossed plants are bulked together to create the cultivar. In
the later scenario, it is unlikely that genetic improvement beyond the recur-
rent parent can be made. From the initial hybridization with a transgenic
donor plant to commercial sales, the process generally takes six years (Table
8.3). Genetic gain during this process is affected by how many plants can be
backcrossed and how many sister-lines can be tested during preliminary and
advanced trials. Breeding costs are further driven up for strict quality
control measures involved with transgenic breeding. Consequently, trans-
genic breeding is much more expensive and less quantitative genetic gain is
accumulated in comparison to strictly conventional cotton breeding
procedures.
Table 8.3 Timeline for commercial transgenic cotton variety development using the back-
cross procedure
Year Activity
1 Backcrossing three generations per year if greenhouses or counter-season nurseries
are used.
2 Final backcross and isolation of homozygous transgenic plants.
3 Seed increase and preliminary yield trials.
4 Seed increase and advanced yield trials.
5 Seed increase and preliminary commercial level testing.
6 Seed sales.
8.8 Integration of New Biotechnologies in Breeding Programs
New biotechnological approaches to transferring genes can complement conven-

tional plant breeding programs (Jauhar 2006). A new era of biotechnology in
cotton breeding has emerged since the first transgenic commercial cotton culti-
vars were released in 1996. Genes conferring resistance to herbicides that kill a
wide range of weeds and genes for resistance to Lepidopteran insects in cotton are
being utilized primarily by private sector cotton breeders. These genes were
inserted into cotton cultivars using biotechnologies including tissue culture,
Agrobacterium-mediated transformation, and/or biolistic transformation.
While using these techniques to develop transgenic cultivars, unique molecular
sequences of DNA (i.e. RFLPs, AFLPs, RAPDs, SSRs, SNPs, etc.) have been
278 S. Hague et al.
identified. These molecular markers potentially are useful in marker-assisted

selection as tools to increase the efficiency of conventional cotton breeding.
Publicly available information about cotton molecular research and other
resources is continuously updated by two databases, CMD (Cotton Microsatel-
lite Database; http://www.cottonmarker.org/; Blenda et al. 2006) and CottonDB
(http://cottondb.org/; Yu et al. 2006).
Tools and techniques of biotechnology can be aimed at improving cotton in
four areas: (1) decrease the time needed to develop cultivars (marker-assisted
selection); (2) genetically modify agronomic characteristics (herbicide and
insect resistance; abiotic stress tolerance; fiber traits); (3) genetically modify
fatty acid profiles (alter type and proportion of fatty acids); and (4) genetically
modify cottonseed meal quality (remove gossypol to extend the use of cotton-
seed to human consumption).
Marker-assisted selection allows breeders to improve efficiency by identify-
ing lines fixed for a given trait early in the line development process. Only plants
carrying the trait of interest are carried forward. Plants without the trait of
interest can be discarded immediately rather than expending time and resources
for phenotypic evaluations. This selection strategy may be useful when
introgressing traits from a related species (Lacape et al. 2003). Zhang et al.
(2003) tested the efficiency of marker-assisted selection to detect quantitative
trait loci (QTL) associated with increased fiber strength. The SSR marker
FSS1130 efficiently detected the homozygous genotype associated with a
major QTL for fiber strength in three segregating populations with the cotton
line 7235 as a parent. The potential of large-scale use of SSR technology is being
evaluated in a diallel study utilizing additional parental lines (Hinze et al. 2007).
Most new biotechnologies to modify agronomic traits in cotton are being
utilized in the breeding programs of private companies. The first transgenic
cotton technology, BXN, was sold in 1995. This allowed plants to be tolerant to
normally lethal doses of bromoxynil herbicides. The following year more
transgenic cotton technologies resistant to the herbicide Roundup1 and Lepi-
doptera insects were released by Monsanto. Insecticide and herbicide resistance
are still the only two transgenic traits marketed as of 2007. New areas of
transgenic research by companies include drought and stress resistance/toler-
ance, salt tolerance, and improved fiber quality. Public institutions continue to
explore biotech solutions to agronomic problems that are less financially
lucrative, but important to stakeholders. Cotton plants transformed with an
endochitinase gene from the Trichoderma fungus have significant resistance to
Rhizoctonia solani and Alternaria alternate (Emani et al. 2003). Tolerance to
Thielaviopsis basicola has also been identified in transgenic seedlings expressing
a synthetic antimicrobial peptide (Rajasekaran et al. 2005).
There is ongoing research into the pathways of lipid synthesis and develop-
ment of transgenic plants to modify the fatty-acid profile of cottonseed oil.
Oleic acid in cottonseed has been increased by suppressing a D-12 fatty acid
desaturase (FAD2) using a mutant allele and inserting this construct into cotton
plants using Agrobacterium-mediated transformation (Chapman et al. 2001).
8 Cotton 279
Other groups have attempted to increase seed oleic acid by antisense

suppression of FAD2 (Liu et al. 2002; Sunilkumar et al. 2005). Successful
research efforts in producing such transformants have not yet been adapted
to commercial cultivars.
Pigment glands are located throughout the cotton plant. These glands
contain gossypol and gossypol precursors that are the greatest quality detri-
ment to cottonseed products. Cotton breeders have been trying for decades to
eliminate the antinutrient gossypol (Muramato 1969; Dilday 1986; Altman
et al. 1987; Vroh et al. 1999). In the 1950s, breeders discovered a mutant that
completely lacked the compound, but the gossypol-less plant was vulnerable to
pests and a commercial failure. To target seeds while keeping defenses intact in
vegetative tissue, molecular biologists began to examine metabolic pathways of
gossypol formation. Attempts to eliminate gossypol in seeds via antisense
suppression of (+) d-cadinene synthase gene methods have been unsuccessful
(Townsend et al. 2005) or have reduced gossypol, but not completely eliminated
it (Martin et al. 2003). Most recently, researchers have achieved a reduction of
gossypol in cottonseed using RNAi to disrupt the gossypol biosynthesis path-
way, specifically by interfering with expression of the d-cadinene synthase gene
during seed development (Sunilkumar et al. 2006). Another approach for
eliminating gossypol is removal of glands that store gossypol in seeds. This
approach is directed toward the dominant Gl2e gene which controls glandless
expression (Kohel and Lee 1984). Research has been initiated to clone the Gl2e
gene and link it in antisense with a seed-specific promoter so that no glands
occur in seeds while they remain unaltered in the rest of the cotton plant (Yu
et al. 2000a,b; Decanini et al. 2001; Kohel et al. 2001).
8.9 Seed Production
High-quality seed production can only occur when cotton bolls are fully
mature and carefully harvested, ginned, and stored. Seeds produced during
the early fruiting stage that are fully mature have a higher protein content, a
more favorable saturated to non-saturated fatty acid ratio, a higher seed
weight, and a greater oil content than seeds from later, less mature fruiting
structures (Conkerton et al. 1993; El-Nockrashy et al. 1976). Generally,
plants managed for high lint production are more likely to produce high-
quality seed. Poor plant nutrition, low-light quality, and drought stress can
each reduce seed weight and quality. High rates of nitrogen and excessive
soil moisture during late-season also can be detrimental to seed quality by
promoting more late maturing bolls. Moreover, delays in harvest that
expose open cotton bolls to moisture is very damaging to seed quality.
Excessive moisture elevates free fatty acid accumulation which is also asso-
ciated with seed embryo mortality (Hoffpauir et al. 1947). Therefore, timely
harvests are crucial to securing the highest quality seed.
280 S. Hague et al.
Insect pests, particularly pink bollworms (Pectinophora gossypiella Saun-

ders) and stinkbugs (Euschistus spp.) can be very damaging to seed quality.
In addition, cottonseed can become infected with Aspergillus flavus and
contain lethal amounts of mycotoxins. Cottonseed infected with A. flavus
usually coincides with seed damaged by pink bollworms. Users of cotton-
seed should regularly test for such contamination. Bt cotton offers excellent
protection against the pink bollworm, and compositional analysis of Boll-
gard II1 cottonseed, a Bt cotton, showed no significant differences in
composition to seed from other non-transgenic cotton cultivars (Hamilton
et al. 2004).
Ginning and storage are important in maintaining seed quality. Gin machin-
ery should be adjusted so that minimal damage to seed coats is incurred.
Ginning practices that chip seed coats promote disease and premature degrada-
tion of the kernel. Storage begins when seed cotton is harvested and packed into
modules for transporting to gins. After ginning, seed is stored at the gin and
later at oil mills or by whole cottonseed users such as dairies. During all storage
steps, airflow is important to maintain low humidity and reduce heat accumu-
lation. These conditions reduce free fatty acid formation and improve the end-
use value of cottonseed.
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Chapter 9
Peanut
Barry L. Tillman and H. Thomas Stalker
9.1 Introduction
Peanut is one of the world’s major sources of vegetable oil. According to United
States Department of Agriculture (USDA) databases, peanut is fifth worldwide
in vegetable oil production among nine major oilseed crops (http://www.fas.us-
da.gov/psdonline/psdHome.aspx). Although peanut is widely viewed as an oil-
seed crop, utilization of peanuts varies greatly from country to country. In some
countries, the majority of production is crushed for oil, whereas in others such
as the United States (US), peanuts are used primarily for food.
Peanut is grown on every continent, but the majority of production occurs in
Asia, Africa, South America, and North America. During the five-year period
1996–2000, China, India, and the United States accounted for almost 70% of
the total annual peanut production globally (Rovoredo and Fletcher 2002). By
country, China accounted for about 39%, India about 25% and the US about
6% of total production. Average pod yield ranged from 0.43 t/ha in Africa to
3.54 t/ha in North America (world average is 1.35 t/ha) (Dwivedi et al. 2007). In
the US, pod yield increased dramatically in the early 1970s, but only slowly
during more recent years (Fig. 9.1). More than 90% of the crop is grown in
developing countries and the majority of the seeds are consumed in the country
of origin. This helps to explain why world trade of peanuts is relatively small.
Historically, only about 5% of peanuts enter the export market, with only three
countries accounting for the majority of these exports.
In the United States, peanut is utilized primarily as a snack food. Industries
that process peanuts and manufacture peanut products require that new culti-
vars deviate from the historical type as little as possible. This constrains genetic
gain for seed size and other agronomic traits because corporations that shell
and process peanuts for food have developed sophisticated manufacturing
techniques and equipment based on a tight range of standards. New cultivars
B.L. Tillman (*)

Agronomy Department, University of Florida, North Florida REC, FL, USA
e-mail: btillman@ufl.edu

288 B.L. Tillman and H.T. Stalker
Fig. 9.1 Five year running average of peanut pod yield in the three primary growing regions of
the US. Legend details: Southeast – Alabama, Florida and Georgia; Southwest – New Mexico,
Oklahoma and Texas; Virginia-Carolina – North Carolina, South Carolina, and Virginia
Source: United States Department of Agriculture.
that do not meet seed industry expectations are less likely to be accepted even
though they may have significant benefits for farmers.
Peanut breeding goals are largely dictated by crop utilization. Although a
majority of world production is crushed for oil, peanut is also heavily utilized as
a food crop. In the 5-year period 1996–2000, nine of the 15 major peanut
producing countries crushed more than 55% of their crop for oil whereas six
used more than 60% for food (Rovoredo and Fletcher 2002). Due to the wide
variation in utilization among countries and regions, peanut breeding goals
may greatly differ. However, some goals such as increasing disease resistance
and yield are common to all breeding programs. Abiotic (drought) and biotic
(mostly fungal and viral diseases) stresses are the major constraints on world
production, and are the focus of nearly all peanut breeding programs.
The domesticated peanut is plagued by many disease and insect pests, with
early leaf spot (Cercospora arachidicola Hori), late leaf spot (Cercosporidium
personatum (Berk. & M.A. Curtis) Deighton), rust (Puccinia arachidis Speg.),
and viruses being widespread wherever the crop is grown. Many other problems
are regional in nature, with Sclerotinia blight (Sclerotinia minor Jagger), white
mold or stem rot (Sclerotium rolfsii Sacc.), Cylindrocladium black rot (Cylin-
drocladium crotalariae (Loos) Bell and Sobers), nematodes (Meloidogyne spp.),
and tomato spotted wilt virus (TSWV) being among the most important dis-
eases in the US. In addition, aflatoxin (caused by Aspergillus spp.) is a major
industry problem that is predominately solved by testing seed samples from
farmer’s fields at buying stations and removing contaminated lots from the
9 Peanut 289
edible market. Allergens also are a major problem for the industry, and because
they are found in the major seed storage proteins, all peanut products (except
purified oils) will cause allergic reactions to susceptible individuals. Much of the
domesticated peanut collection has been screened for resistance to the leaf spots
and rust (see Holbrook and Stalker 2003 for review), and although moderate
levels of resistance have been identified, extremely high levels of resistance
apparently do not exist in the germplasm collection. Variation has also been
observed for reaction to viruses, such as TSWV, but the resistance is due to
unknown effects of the environment in the field rather than to the virus per se
(see Holbrook and Stalker 2003). Resistances to many of the most important
disease and insect pathogens of peanut have been identified in other species of
the genus, including members of section Arachis (to which the domesticated
peanut belongs), and thus the potential for genetic improvement of A. hypogaea
through interspecific hybridization exists (Stalker and Simpson 1995).
Although peanut originated in South America, these countries produce only
a small fraction of the world’s crop. Argentina is the largest peanut producer in
South America, followed by Brazil. South America is rich in peanut genetic
resources and both wild and cultivated genotypes from that continent have
contributed to improved cultivars in the United States and elsewhere.
Peanut is a low-growing annual or perennial plant that is distinguished from

most other species by producing aerial flowers, but fruiting below the soil level.
Arachis belongs to the family Fabaceae, tribe Aeschynnomeneae, subtribe Sty-
lanthinae. Arachis hypogaea L. is the only domesticated species in the genus, and
Krapovickas (1969) concluded that A. hypogaea var. hypogaea is likely the most
ancient type because it has similar branching patterns as wild species, no
compound florets, and a prostrate growth habit. The primary center of diversity
for the species is the Chaco region between southern Bolivia and northwest
Argentina (Gregory and Gregory 1979). The peanut is now grown in most
tropical to subtropical regions of the world, with more than 90% of the world’s
production in Asia and Africa. Peanut seeds contain 36–54% oil, 16–36%
protein, and 10–20% carbohydrates (Knauft and Ozias-Akins 1995). It also is a
good source of minerals (Ca, Mg, P, and K) and vitamins E, K, and B1 (Savage
and Keenan 1994). At least two additional species have been cultivated for their
edible seeds (A. villosulicarpa Hoehne and A. stenosperma Krapov. and W.C.
Gregory), a few species are cultivated as a forage (A. glabrata Benth. and A. pintoi
Krapov. and W.C. Gregory), and A. repens Handro is grown as a ground cover
for lawns in Central and South America (Stalker and Simpson 1995).
The center of origin for the genus Arachis is the Mato Grosso area of Brazil,
but species evolved over a wide range of habitats in South America (Gregory
et al. 1980). Molecular data indicate that the center of genetic variation also is
the Mato Grosso area of Brazil to eastern Bolivia (Stalker et al. 1994). Because
the most ancient species are believed to have tuberoid roots, tuberiform hypo-
cotyls, or rhizomes, the first species are thought to be from highland areas, with
subsequent distribution by water to lower elevations. Eighty species have been
described (Krapovickas and Gregory 1994; Valls and Simpson 2005) which
have been divided in to nine sections based on morphology and cross-compat-
ibility relationships. Based on cross-compatibility data, Smartt and Stalker
(1982) and Stalker (1991) concluded that genomic groups have evolved in
the genus which mostly follow sectional designations (Am – Ambinervosae,
T – Triseminalae, C – Caulorhizae, EX – Extranervosae, and E – Erectoides,
R – Rhizomatosae, and A, B and D – Arachis). Smartt and Stalker (1982) also
proposed that the A and B genomes of section Arachis may be an A1 and A2
rather than being truly different based on chromosome pairing relationships.
The species of different sections have overlapping distributions in many areas.
Hybrids between species in different sections are difficult to produce and are
usually sterile, while intrasectional hybrids can be fertile if they have similar
genomic make-up (Stalker et al. 1991). Most species in the genus are diploid
(2n = 2x = 20), but tetraploids (2n = 4x = 40) exist in sections Arachis and
Rhizomatosae, and several species in section Arachis are aneuploid (2n = 2x =
18) (Lavia 2000). Polyploidy is believed to have evolved independently in
sections Arachis and Rhizomatosae (Smartt and Stalker 1982), and likely inde-
pendently more than once in section Rhizomatosae (Nelson et al. 2006).
The domesticated peanut (A. hypogaea) evolved from two diploid species of
section Arachis approximately 3500 years ago in the southern Bolivia to north-
ern Argentina region of South America (Gregory et al. 1973). Secondary centers
of diversity developed in South America and tertiary centers in Africa (Smartt
and Stalker 1982). Domesticated peanut is taxonomically a member of section
Arachis, along with 25 other diploid (2n = 2x = 20), four aneuploid (2n = 2x =
18) and one tetraploid (2n = 4x = 40) species. Species in this section have
evolved into three genomic groups, with most species having an A genome, six
species having a B genome, and A. glandulifera Stalker a D genome (Holbrook
and Stalker 2003; Valls and Simpson 2005). The domesticated peanut is
believed to be an allopolyploid with AABB genomes because it has only one
small chromosome pair in somatic cells and most meiotic cells have 20 biva-
lents, although multivalents can occur. Kochert et al. (1991) supported this
conclusion because they observed multiple bands on RFLP gels for A. hypo-
gaea, but only single bands for diploid species. Many species have been pro-
posed as possible progenitors, but the most likely candidates are A. duranensis
Krapov. and W.C. Gregory (A-genome) and A. ipaensis Krapov. and W.C.
Gregory (B-genome) (Kochert et al. 1996). Arachis duranensis is believed to be
the female parent of the ancestral hybrid based on analyses of cytoplasmic genes
(Hilu and Stalker 1995).
As compared to wild species of the genus, domestication of A. hypogaea led
to an upright growth habit, shorter branches, suppressed hypanthium length,
stronger and shorter pegs, and pods with the internode between seeds
9 Peanut 291
suppressed (Stalker and Simpson 1995). The most ancient A. hypogaea types
have alternating inflorescences, main stems without flowers, and flowers that
are simple (vs. compound inflorescences), prostrate growth habits, are late
maturing, with hairy leaves, 2-seeded pods with a beak, small seeds with a
long dormancy period, and long lateral branches (Stalker and Simpson 1995).
The species A. hypogaea has two subspecies and six botanical varieties
(Table 9.1). The subspecies are separated morphologically based on pre-
sence or absence of flowers on the main stem and regularly alternating
vegetative and reproductive nodes on branches. Although accessions of A.
hypogaea are morphologically variable, relatively little molecular variation
has been observed. This led Kochert et al. (1996) to conclude that there
has been little introgression from related species into the domesticated
peanut since its origin. In addition to morphological and genetic variation,
Table 9.1 Subspecies and varieties of A. hypogaea1

Variety Market type S.A. location Characteristics
Subspecies hypogaea
hypogea Bolivia, Amazon No floral axes on main stem;
alternating pairs of floral and
reproductive axes on branches;
branches short; less hairy
Virgina Less hairy; large seeded
Runner Less hairy; small seeded
hirsuta Peruvian Peru More hairy
runner
Subspecis fastigiata
fastigiata Floral axes on main stem;
alternating pairs of floral and
vegetative axes on branches
Valencia Brazil Little branched; curved branches
Guaranian
Goias
Minas Gerais
Paraguay
Peru
Uruguay
peruviana Peru Less hairy, deep pod reticulation
N.W. Bolivia
aequtoriana Ecuador Very hairy; deep pod reticulation;
purple stems; more branched;
erect
vulgaris Spanish Brazil More branched; upright branches
Goias
Minas Gerais
Paraguay
Uruguay
1
After Stalker and Simpson (1995).
Stalker and Dalmacio (1986) reported at least five translocation types in

different varieties of A. hypogaea.
9.3 Varietal Groups
Two botanical groups of cultivated peanut (A. hypogaea) are defined for the
presence or absence of flowers on the main stem (Knauft et al. 1987). The sub-
species A. hypogaea subsp. hypogaea flowers only on the lateral branches
whereas A. hypogaea subsp. fastigiata flowers on both the lateral branches
and main stem. Two varieties are defined within each of these sub-species;
var. hypogaea and hirsuta are of sub-species hypogaea and fastigiata and vul-
garis are of sub-species fastigiata (Simpson and Coffelt 1997).
Similar to the botanical classifications, peanut is divided into market types
which have economic consequences in the United States. The market types
called ‘runner’ and ‘virginia’ are within variety hypogaea. The ‘valencia’ market
type is in the variety fastigiata and the ‘spanish’ market types are within the
vulgaris variety. There are no commercial types of the variety hirsuta grown in
the United States, but plant introductions from Mexico have proven valuable in
breeding programs for their resistance to TSWV.
A pedigree analysis of the cultivars grown in the US shows that most of them
have ancestors belonging to both A. hypogaea subspecies (Isleib and Wynne
1992). Plant introductions have been of great importance in peanut, in large
part for resistance to Sclerotinia blight, root-knot nematode, and TSWV (Isleib
et al. 2001).
9.3.1 Market Types in the United States
Of the four market types of peanut (runner, virginia, spanish, and valencia)
produced in the US, runner and virginia types are the most widely grown. Both
usually have two seeds per pod and the USDA has created standards that define
these two market types on the basis of pod width. Pods that are wider than
13.5 mm are defined as ‘virginia’ pods. Genotypes that have less than 40%
‘virginia’ pods are classified as runner market types and those with at least 40%
are defined as virginia market types. The percentage of virginia pods can be
greatly influenced by environment. Some genotypes can have more than 40%
virginia pods in one environment and less than 40% in another, which would
technically classify them as different market types. In reality, the United States
industry definition of the acceptable range of pod and seed size for runner and
virginia market types is stricter than the USDA definition. In addition to the
percentage of virginia-type pods, seed size is important in both runner and
virginia market types.
9 Peanut 293
In the US, most commonly grown runner market type cultivars have between
10 and 20% virginia pods although the correlation between virginia pods and
seed size is loose. There are three primary seed size classes for runner peanuts.
Seed that ride a screen with 8.3 mm by 25.4 mm slots are called jumbo seed,
those that fall through, but ride a screen with 7.1 mm by 19.1 mm slots are called
medium seed and those that fall through both larger screens but ride a screen
with 6.4 mm by 19.1 mm slots are called number one seed. Seed that fall through
all three screens are called other kernels. Seed that split, but are otherwise
normal, are called sound splits.
In the United States, the price that a farmer receives for a certain mass of
runner type peanuts is adjusted based on the percentage of total sound mature
kernels or ‘grade’ (TSMK). The TSMK is total percentage of the jumbo,
medium, number one and sound split seeds resulting from shelling a certain
mass of peanut pods (Davidson et al. 1982). Above about 74% TSMK, farmers
receive a premium price, but the value is discounted below the threshold.
In addition to the runner and virginia types, two other market types are
grown in the US. The spanish market type is characterized by small seed and
early relative maturity when compared to the runner types. Along with runner
types, spanish types are used primarily for peanut butter, oil and candy. The
valencia market type peanut has more than two seeds per pod and is used
primarily for in-shell roasting and boiling. Virginia types are used for in-shell
roasting and salted or cocktail peanuts.
Several review articles have been published that summarize genetic resources
of the domesticated peanut and related Arachis species (Isleib and Wynne
1992; Singh and Simpson 1994; Stalker and Simpson 1995; Dwivedi et al.
2003; and Holbrook and Stalker 2003), so only a brief review will be pre-
sented. The A. hypogaea accessions are maintained in large germplasm collec-
tions by the USDA (more than 8,000 accessions) (Holbrook 2001) and by the
International Crops Research Institute for the Semi-Arid Tropics (ICRI-
SAT), which has an international mandate for peanut improvement (14,966
accessions) (Upadhyaya et al. 2001). Duplication of accessions, either within
or between the two collections, has not been addressed. Descriptors for pea-
nut have been published by the International Board for Plant Genetic
Resources (IBPGR) and ICRISAT (1992) and by the USDA (Pittman
1995). Germplasm preservation of the domesticated peanut is relatively
straightforward because plants are self-pollinating (although out-crossing
does occur), with the major constraint to long term storage being sufficient
low temperature and humidity facilities because peanut is a large-seeded crop.
Most of the germplasm collection at ICRISAT has been evaluated for biotic
and abiotic stresses commonly found in the semi-arid tropics and the
information has been summarized by Dwivedi et al. (2007). Although large

numbers of accessions have been evaluated for agronomically useful traits in
the USDA and ICRISAT collections, relatively few accessions have been
utilized by breeders for cultivar development in the US (Isleib et al. 2001)
or at ICRISAT (Dwivedi et al. 2007).
Core collections were developed by Holbrook et al. (1993) who subdi-
vided the larger US collection into 831 accessions based on six morpholo-
gical traits. Upadhyaya et al. (2003) developed a second core collection
consisting of 1,704 accessions from the ICRISAT collection. Upadhyaya
et al. (2002) also developed a minicore consisting of 184 accessions based
on agronomic and quality traits among the core accessions; and Holbrook
and Dong (2003) later developed a second minicore with 111 entries in the
US based upon morphological traits. Germplasm evaluations of the core
accessions at ICRISAT identified accessions with early maturity (Upad-
hyaya et al. 2006), tolerance to low temperatures (Upadhyaya et al. 2001),
and drought tolerance (Upadhyaya et al. 2005). Evaluations of US core
collections have identified new sources of resistance for Cylindrocladium
black rot and early leaf spot (Isleib et al. 1995), TSWV (Anderson et al.
1996), root-knot nematode (M. arenaria (Neal) Chitwood) and preharvest
aflatoxin contamination (Holbrook et al. 1998), rhizoctonia limb rot (Rhizoc-
tonia solanii Kuhn) (Franke et al. 1999), and Sclerotinia blight and pepper
spot (Lfeptosphaerulina crassiasca (Sechet) Jackson and Bell) (Damicone
et al. 2003).
In addition to A. hypogaea collections, more than 1,300 Arachis species
accessions have been collected (Stalker et al. 2002c), with about 800 Arachis
entries being maintained by the USDA (Stalker and Simpson 1995). Preserva-
tion of wild Arachis species is difficult for most accessions because the long,
fragile pegs break during harvest and soil must be sifted to recover pods. Stalker
and Simpson (1995) reported that nearly 25% of the species from which seed
can be obtained under nursery conditions have fewer than 50 seeds in storage.
Additionally, at least 25% of the Arachis species accessions in germplasm
nurseries are maintained vegetatively because of their poor seed production
under cultivation. A large number of disease, insect, and other agronomically
useful traits are present in accessions of Arachis species, which makes them
potentially valuable genetic resources for crop improvement (Stalker and Moss
1987; Stalker and Simpson 1995).
Peanut breeding in the United States began in the late 1920s (Knauft et al. 1987;
Gorbet 1999). Since then, peanut cultivars have changed and improved drama-
tically. Incremental improvements in cultivars have occurred over time, but
several major achievements stand out, including release of a single dominant
cultivar, changes in oil chemistry, and improved disease resistance.
9 Peanut 295
9.5.1 Florunner Cultivar
In 1969, the cultivar Florunner was released by the University of Florida

(Norden et al. 1969). Tests showed that the pod yield of Florunner was about
18% greater than Early Runner, the dominant runner cultivar of that time. By
1974, Florunner occupied more than 90% of the acreage in the southeastern
US, and for almost 20 years it remained the dominant runner type cultivar by a
large margin (Gorbet 1999). The seed, pod, and grading characteristics of
Florunner were so good that it quickly became the standard, not only for
peanut farmers, but also for shellers and manufacturers, and remains the
standard against which shellers and manufacturers measure new cultivars. To
a large extent, Florunner is the reason that the US industry has maintained
high-quality products.
In the mid 1990s, TSWV became endemic in the Southeast and eventually
eliminated production of Florunner and other susceptible cultivars. However,
the cultivar Flavorunner 458 is a mutation of Florunner with elevated levels of
oleic acid (Horn et al. 1997). It is virtually indistinguishable from Florunner
otherwise and is widely grown in Texas where TSWV is not problematic.
Florunner was introduced into Argentina during the early 1980s and was
grown on about 80% of their acreage for about 10 years. Later, a selection from
Florunner called Florman was released as a cultivar in Argentina (Casanoves
et al. 2005; J. Baldessari 2007, personal communication) and was grown on
about 80% of the acreage by the late 1990s. Florunner has also been used as a
parent in China. The cultivar Lu Hua 15 has Florunner in its ancestry and has
been widely grown in Shandong Province, China (Xue and Isleib 2002).
Florunner had a dramatic and long lasting impact on the peanut industry in
the US and Argentina. Twelve cultivars released in the US are direct descen-
dants of Florunner (Isleib et al. 2001) and no other runner type cultivar has had
such a significant impact on the peanut industry.
9.5.2 High Oleic Acid Content

The fatty acid profile of normal peanut is similar to other food nuts such as
hazelnut, walnut, almond, and macadamia (Maguire et al. 2004). Compared to
soybean, the major oilseed crop in the US, typical peanut oil has more oleic
acid, less linoleic acid and no linolenic acid (White 2000). Partly because of its
fatty acid profile, peanut oil is considered to be a premium oil and is desirable for
cooking and salad oil (McWatters and Cherry 1982). Oleic and linoleic acids make
up about 80% of the total fatty acids in peanut oil and are inversely correlated
(Worthington and Hammons 1977). Peanut genotypes differ in the amount
of each of these fatty acids, but most typical peanut cultivars contain 45–50%
oleic acid and 30–40% linoleic acid. Prior to 1987, the highest level of oleic
acid found in a peanut genotype was around 69% with a corresponding level of
linoleic acid of about 15% (Worthington and Hammons 1977; Treadwell et al.
1983). In 1987, Norden et al. reported discovery of two experimental peanut lines
that contained almost 80% oleic acid and only 2% linoleic acid. Later work
showed that the trait is conditioned by two recessive alleles (Moore and Knauft
1989). One of the alleles appears to be relatively common in runner and virginia
germplasm, but the other is extremely rare (Knauft et al. 1993). Normal oleic acid
content is incompletely dominant to the high oleic trait so that heterozygous
genotypes can be distinguished from either of the homozygous genotypes (Isleib
et al. 2006c).
The primary benefit of high oleic peanuts is dramatically improved product
storage life. This has been demonstrated in whole peanuts as well as for peanut
oil. A common measure of product stability is the peroxide value which is
associated with the rancidity that causes off-flavors in peanuts and peanut
products. In a study of roasted, in-shell virginia type peanuts, normal oleic
acid peanuts reached an unacceptable peroxide value in only four weeks com-
pared to 32 weeks for the high oleic peanuts (Mozingo et al. 2004). Similar
results were obtained with peanuts that were salted in-shell. High oleic peanut
oil also proved to be more resistant to oxidation as measured by peroxide
values. Peroxide values of the normal oleic acid peanut oil began to rise rapidly
after only 47 h of heating whereas the peroxide value of the high oleic peanut oil
did not begin to rise until over 650 h (O’Keefe et al. 1993).
The first high oleic cultivar released in the US was a runner type named
SunOleic 95R (Gorbet and Knauft 1997). Since that time, the trait has been
incorporated into virginia and spanish types (Isleib et al. 2006b; Simpson
et al. 2003a). Although several cultivars with the high oleic trait are in
commercial production in the US, as of 2005 they occupied less than 20%
of total acreage. Adoption of high oleic cultivars has been slowed partly
because yield performance of high oleic cultivars lagged slightly behind the
traditional types and because the market has had difficulty determining and/
or capturing its economic value. Some manufacturers who have marketed
high oleic peanuts exclusively have reported improved product performance
and consumer acceptance. If high oleic cultivars become dominant in the
marketplace it is likely that manufacturers will realize the benefits on a large
scale and high oleic peanuts would become preferred over normal oleic
peanuts.
9.5.3 Resistance to Leaf Spots, Root-Knot Nematode, and Spotted

Wilt
In the Southeastern US, controlling diseases and nematodes in peanut accounts

for about 20% of the total variable costs. In areas where pesticides are not
available, or are too expensive for subsistence farmers to purchase, diseases and
pests substantially reduce yield. Cultivar resistance has the potential to improve
9 Peanut 297
production in situations of limited crop inputs and reduce costs where pesticides
are used. The diseases that breeders target are sometimes localized to a parti-
cular environment, but there are diseases and pests that have widespread
importance, such as leaf spots and root knot nematodes.
On a worldwide basis, leaf spot diseases caused by Cercospora arachidicola
Hori and Cercosporidium personatum (Burk. & Curt.) are arguably the most
widespread, devastating and costly to control of the many diseases that afflict
peanut (Shokes and Culbreath 1997). Yield losses of 50% are common when
fungicides are not applied, and losses can be 70% or more (Shokes and Cul-
breath 1997). Historically, control is achieved by application of fungicides
because there were no cultivars with sufficient resistance. The first leaf spot
resistant cultivar in the United States developed from the University of Florida
program was Southern Runner (Gorbet et al. 1987). Its leaf spot resistance was
derived from PI 203396. Since D.W. Gorbet at the University of Florida began
an intensive effort to breed peanut for resistance to leaf spots, several cultivars
with rate limiting, partial resistance have been released (Chiteka et al. 1988a,b).
Research has shown that fungicide applications targeting leaf spot can be
reduced by half on these cultivars with little or no reduction in pod yield and
grade (Gorbet et al. 1990). The same study showed that pod yield of resistant
genotypes was double that of susceptible lines when no fungicides were applied.
These cultivars offer the potential to significantly reduce fungicide cost, but
they have not been widely accepted by the industry. Seed quality has been an
overarching problem with leaf spot-resistant lines and commercial success of
cultivars with the highest levels of resistance has been limited (Morton 2007;
Morton et al. 2006). In the future, utilization of the significantly higher levels of
leaf spot resistance in Arachis species than found in A. hypogaea may aid peanut
breeders in developing new cultivars. Germplasm lines have been released with
very high levels of leaf spot resistance derived from A. cardenasii (Stalker et al.
2002b).
Similar to leaf spots, the root-knot nematode species that attack peanut
(Meloidogyne arenaria (Neal) Chitwood, M. hapla Chitwood, and M. javanica
(Treub.) Chitwood) are distributed worldwide (Kokalis-Burelle and Rodriguez-
Kabana 1997). Yield losses in severely infested fields can be as high as 90%.
Since root-knot nematodes are soil borne, rotation to a non-host crop can be an
effective control measure. However, economics of rotational crops in some
peanut growing areas prevent adequate crop rotation for many farmers.
Continuous peanut culture has resulted in fields that are heavily populated by
root-knot nematodes and a need for resistant cultivars has emerged. Moderate
levels of M. arenaria resistance have been identified in A. hypogaea (Minton and
Hammons 1975; Holbrook and Noe 1992) whereas several Arachis spp. have
high levels of resistance to root-knot nematodes (Nelson et al. 1989; Holbrook
and Noe 1990). Subsequent interspecific hybrids between cultivated peanut and
accessions in section Arachis were highly resistant to M. arenaria and M.
javanica (Nelson et al. 1990; Starr et al. 1990, 1995; Stalker et al. 2002a). Genetic
control is believed to be monogenic and dominant in most germplasm, but a
second dominant gene has been reported (Choi et al. 1999). The cultivars
COAN (Simpson and Starr 2001) and NemaTAM (Simpson et al. 2003b)
contain the nematode resistance gene, but since they are essentially derived
from Florunner, they have limited production because they are highly suscep-
tible to TSWV. Another breeding line (C724-19-15 from the USDA in Georgia)
that contains this gene for nematode resistance and has good TSWV resistance
has been released as ‘Tifguard’ (C.C. Holbrook 2007, personal communication;
Holbrook et al. 2007). Cultivars with both nematode and TSWV resistance
would be beneficial to growers in the Southeastern US.
A third, very destructive disease in the US is spotted wilt caused by tomato
spotted wilt virus (genus Tospovirus; family Bunyaviridae) (Culbreath et al. 2003).
While several production factors have been shown to reduce the risk of crop loss
from spotted wilt, cultivar resistance is the most important factor (Brown et al. 2005).
When spotted wilt severity began to rise significantly in the southeastern US during
the middle 1990s, all widely grown cultivars were very susceptible and spotted wilt
caused significant crop losses in Texas and the Southeast (Culbreath et al. 2003).
Researchers in Texas first discovered that the cultivar Southern Runner had some
resistance to spotted wilt (Black 1991), which is thought to have come from PI
203396. The cultivar Georgia Green, a cross between Sunbelt Runner and Southern
Runner, was released just before the height of the spotted wilt epidemic in the
southeastern US (Branch 1996). It had inherited a moderate level of field resistance
to spotted wilt from Southern Runner and quickly became the dominant runner
market-type cultivar grown in the southeastern US. Without spotted wilt resistance
in Georgia Green, the peanut industry in the Southeast would have been crippled.
9.6 Goals of Peanut Breeding
The goals of peanut breeding revolve largely around the requirements of the
various ‘customers’ of peanuts and peanut products including farmers, shellers/
seedsmen, manufacturers, and consumers. Specific goals have changed little
since the reviews by Norden (1973), Knauft et al. (1987), Norden et al. (1982),
Knauft and Ozias-Akins (1995), and Holbrook and Stalker (2003), so a brief
summary will be presented in this chapter. Although some goals are common to
most peanut breeding programs, the different market types of peanut have
unique problems that must be addressed by peanut breeders at different institu-
tions. Goals that are common to all market types include pod yield, market
grade, high oleic oil, and disease resistance.
9.6.1 Goals for the Farmer

Peanut farmers provide much of the operational funding for peanut breeding in
the US through state and federal government funding mechanisms and royalties
9 Peanut 299
paid on seed sales. Therefore, much of the work in peanut breeding is focused to
their needs. Peanuts are sold on a tonnage basis, so the per acre pod yield and
grade (TSMK) are the two primary factors that determine crop value for the
farmer. Therefore, all new cultivars must demonstrate improvement in one or
both of those areas. Pod yield of peanut has risen significantly since the 1940s
(Fig. 9.1). The contribution of breeding to pod yield improvement is significant,
but few studies have documented it. The genetic contribution to yield in virginia
market types was estimated to be 14.7 kg/ha/year from 1944 to 1985 (Mozingo
et al. 1987), but similar studies have not been published for the other market
types. However, the sharp increase in yields in the southeastern US can be partly
attributed to the Florunner cultivar as described in Section 9.5.1. Tests showed
that the yield of Florunner was about 18% greater than the dominant runner
market type cultivars at the time of its release in 1969 (Norden et al. 1969).
In addition to pod yield and grade, resistance to diseases and pests is a
breeding goal focused to the needs of the peanut farmer. In the US, peanut
diseases vary regionally although some are common to all regions. Diseases of
regional importance include white mold, Cylindrocladium black rot, spotted
wilt, and Sclerotinia blight. Where these diseases are important, breeders are
actively screening germplasm and breeding populations for resistance. Diseases
and pests of national and international importance include leaf spots, root knot
nematode and spotted wilt.
Breeders throughout the US and other parts of the world are working to
develop peanut cultivars with resistance to the leaf spot diseases described in
Section 9.5.3. Although cultivars with partial resistance to leaf spot are avail-
able in the US, production has been limited. The major limiting factor is seed
vigor, which has been generally poor when these cultivars are grown and
handled in the commercial seed chain (Morton et al. 2006). Unfortunately,
the physiological or chemical cause of reduced seed vigor is not known. A
second factor that limits production of leaf spot resistant cultivars is relative
maturity. As a group, the leaf spot resistant cultivars are about 14 days later
maturing than the cultivars that are most commonly grown. Producers prefer
earlier maturing cultivars partly because of the logistics of planting and harvest-
ing other crops planted in rotation with peanut and to avoid freeze damage to
seeds. Similarly, the cost and risk associated with an additional two weeks that
the crop is in the field limits their appeal.
9.6.2 Goals for the Seed Producer/Sheller
Commercial companies that purchase and shell peanuts are also the primary
seed producers and marketers in the US. Their customers are both peanut
farmers and companies that manufacture peanut products. As a seed producer
and marketer, traits that are important to farmers are important to them as
well. Cultivars with the best yield and grade are favored by peanut farmers.
Where disease limits either of these components, disease resistant cultivars

become very important, for example, with spotted wilt when the cultivar
Georgia Green was introduced (see Section 9.5.3).
As a peanut sheller, traits that affect the shelling process as well as those that
affect the merchantability of the shelled peanuts are important. Peanut shelling
companies want cultivars with uniform pod size that shell easily. Uniformity in
pod and seed size minimizes segregation in storage warehouses and minimizes
modifications in shelling machinery and processes. Cultivars with uniform seed
and/or pod size are also desired by their other customers, the manufacturers of
peanut products.
9.6.3 Goals for the Manufacturer and Consumer
In the US and other countries where peanut is used primarily for food, manu-
facturers and consumers demand high quality, nutritious peanuts and peanut
products. In areas where peanut is used primarily for oil, traits needed for food
use are not as important. Peanut flavor is cited by manufacturers as the most
important attribute for peanut consumers. Traditionally, the runner and span-
ish market-types have had the best flavor profiles. Flavor is heritable, but
estimates of broad sense heritability are relatively low at about 0.01–0.31
(Pattee et al. 1993; Isleib et al. 2006a). Due to the costly and time consuming
process of taste panels evaluating flavor attributes, most breeders conduct
flavor evaluations on only a few advanced breeding lines each year. This can
lead to release of agronomically superior cultivars with slightly inferior flavor
profiles (Isleib et al. 2000). However, breeding lines and cultivars with improve-
ments in flavor attributes are being identified within all market types. Recently,
researchers from North Carolina State University have identified virginia mar-
ket-type breeding lines with flavor profiles similar to runner market-types
(Pattee et al. 2007). One factor in maintaining flavor of peanut and peanut
products is consistency of roasting. Because seed size contributes to roast
variability, manufacturers have communicated their desire to have about 50%
of the shelled peanut stock from runner market type cultivars in the medium
category. If the raw peanut product is consistent, then the variability in the final
roasted product should be more consistent as well. Interestingly, this require-
ment is not shared by all manufacturers. Peanut butter manufacturers are the
primary benefactor of an abundance of medium sized seed. Because they utilize
about 50% of the peanuts produced in the US, breeders of runner market-type
peanuts pay close attention to the percentage of medium seed during the
breeding process. Some manufacturers of confectionary products prefer the
smaller spanish market-type seeds for candy bars. Others prefer a variable raw
product so that the finished product does not look artificial. Still others want
large virginia market-type peanuts or the jumbo seeds from runner market-
types for whole roasted and salted peanuts. Virginia market-type peanuts are
9 Peanut 301
primarily marketed in-shell. As such, the color and texture of the pod is
important.
Manufacturers also want cultivars with high oleic acid content. As described
in Section 9.5.2, high oleic peanuts offer several advantages over those with
normal oleic acid. Among these advantages is extended shelf life. Since flavor is
such an important attribute for peanut consumers, manufacturers realize that
high oleic cultivars could help maintain peanut flavor and, therefore, enhance
consumer acceptance.
Breeding methods used by peanut breeders are those common to other self-
pollinated crops and include pedigree selection, single seed descent, and mass
selection. In the US, many breeders practice single seed descent whereas others
use the pedigree method or a combination of both. Some cultivars have been
developed by mutation such as Flavorunner 458 (Horn et al. 1997), but muta-
tion breeding is not common. Breeders have employed backcrossing to intro-
gress nematode resistance (Simpson et al. 2003a, b) and the high oleic trait
(Isleib et al. 2006b). Crossing is usually conducted in greenhouses and general
methods have been previously described (Norden 1980). Several reviews have
been written that thoroughly describe peanut breeding methods (Knauft and
Ozias-Akins 1995; Knauft et al. 1987; Norden et al. 1982; Norden 1973). Since
the basic methodology has not changed or has changed very little, this section
will focus on the unique aspects of peanut breeding that arise from the unique-
ness of the peanut plant and peanut as a crop.
Unlike any other common oilseed crops, the peanut plant bears fruit under-
ground. This phenomenon creates several challenges in the breeding process
regardless of the breeder’s preferred method of selection. Perhaps the most
difficult problem is determining maturity. Because peanut has an indeterminate
flowering habit and pods are underground, clues about seed maturity are not
readily visible as the crop matures. With thousands of plots to harvest and
hundreds of genotypes varying in maturity, precise prediction of optimum
maturity is very difficult. The most practical technique for determining matur-
ity is to scrape away the outer layer of the pod to reveal inner layers (Sanders
et al. 1982; Knauft et al. 1987; Baldwin 1990). As the peanut pod matures, the
subcutaneous layers change color from cream to orange, then brown and finally
black. Most breeding programs use the pod scrape method to determine matur-
ity of a few known genotypes and then set a harvest schedule accordingly.
Testing every genotype is usually not feasible.
Another problem presented by the geocarpic nature of peanut is one of
harvest logistics and high moisture levels. Whereas the harvest operation of
most oilseed crops is a single step, peanut harvest requires several. First, the
peanut plants must be dug from the soil. Since the plant is still living at the time
of digging, the green foliage must be allowed to dry for several days before
combining, during which time pods and seeds also begin to lose moisture.
Second, the pods must be threshed from the plants in an operation similar to
the harvest of other typical oilseed crops, and then the seed is dried by forced,
heated air. This harvest process requires considerably more manpower, time,
and energy than harvest of other oilseed crops. Similar to other members of the
pea (Fabaceae) family, peanut seeds are enclosed in a pod, but in the case of
peanut, the seed are not separated from the pod in the harvest process. Seed
must be separated from the pods during a shelling and cleaning operation after
they are sold by the farmer. Because of the uniqueness of the peanut plant, some
individuals have suggested that breeding peanut is more similar to breeding a
vegetable crop than a traditional row crop like soybean, canola, or sunflower.
Another issue that makes breeding peanut a challenge is the size of the seed
and the sowing density. Peanut seed ranges in size from one half gram per seed
to over one gram per seed. There are about 1,500 seeds/kg of a typical runner
market-type cultivar but this can vary considerably among cultivars. In the
southeastern US, farmers plant about 125 kg/ha of runner market types and
harvest about 2,100 kg/ha of seed for seed increase ratio of 15:1 to 20:1. In
contrast, soybean seed are planted at about 73 kg/ha with a seed yield of around
2,700 kg/ha for a seed increase ratio of 30:1 to 40:1. Thus the seed increase ratio
for peanut is about half that of soybean. Because of this, peanut breeders must
devote considerable resources to producing seed for planting tests, especially
tests planted at several locations.
Molecular technologies have the potential for greatly increasing breeding effi-
ciency of peanut, but marker systems are needed to enhance selection for
desired traits. Because resistance to many of the disease and insect pests of
peanut is difficult to select in the field or greenhouse, molecular technologies
have the greatest potential for solving these problems. For example, selection
for resistance to TSWV, Sclerotinia blight, white mold, Cylindrocladium black
rot, and leaf spots could be greatly enhanced by utilizing marker technologies.
In particular, molecular markers could allow breeders to select multiple genes
when traits have complex inheritance and multiple mechanisms of host-pathogen
resistance (for example in the leaf spots). To date, molecular technologies have
played a minor role in peanut breeding programs, with nematode resistance being
the only trait where it has been effectively applied.
In addition to traits that are important for producers, consumer-oriented
characters, such as eliminating toxins and allergens, are of great importance to
the peanut industry because most of the US grown peanuts are consumed by
humans. Little progress has been made through traditional breeding in this
area, and applying molecular technologies has the potential for solving several
9 Peanut 303
highly complex problems. Genetic enhancement of several health-related traits

also would expand markets, for example increased folate levels, additional
flavanoid production, increased vitamin E, the introduction of Vitamin A
into the seed (from other species), and modification of proteins to make them
more digestible. To better coordinate molecular genetic research efforts in
peanut, a strategic plan was prepared in 2004 by a group of peanut breeders
and geneticists in consultation with industry representatives to set priorities in
six areas, including (1) genetic tools and breeding methods, (2) plant transfor-
mation technology, (3) genomic sequencing and gene discovery, (4) functional
genomics and proteomics, (5) immunology of peanut proteins in model systems,
and (6) bioinformatics (Wilson 2006).
9.8.1 Marker Development in Peanut
Molecular marker development in peanut has been a slow process in large part
because of the low levels of polymorphism identified among A. hypogaea
accessions (Halward et al. 1991; Kochert et al. 1991). Isozymes, Restriction
Fragment Length Polymorphisms (RFLPs), Randomly Amplified Poly-
morphic DNAs (RAPDs), Amplified Fragment Length Polymorphisms
(AFLPs), Cleaved Amplified Polymorphism (CAP), Single Sequence Repeats
(SSR), and Reverse Transcriptase Polymerase Chain Reaction (RTPCR) have
all proved ineffective for producing sufficient numbers of polymorphisms to
create a saturated molecular map in the domesticated peanut (Paterson et al.
2004). For example, He and Prakash (1997) used 28 primer pairs to generate 111
AFLPs for DNA markers in A. hypogaea, with about 3% of the primers being
polymorphic. AFLPs were the first molecular marker system used in peanut to
separate botanical varieties of A. hypogaea (He and Prakash 2001) and the
marker system was later used to differentiate closely related peanut cultivars
(Herselman 2003). Hopkins et al. (1999) isolated the first SSR markers in
peanut, and He et al. (2003) reported that microsatellites developed from
SSRs are more variable than other types of markers. They identified 19 poly-
morphic markers among A. hypogaea genotypes. Many hundreds of SSR
makers have been developed during recent years (Jayashree et al. 2005; Luo
et al. 2005; Ma et al. 2007), with less than 30% being polymorphic among A.
hypogaea lines (Ferguson et al. 2004; Moretzsohn et al. 2004; He et al. 2005).
SSRs can be used to separate cultivars (Moretzsohn et al. 2005) and this marker
system holds great potential for developing useful markers to improve selection
efficiency for traits in peanut breeding.
To date, only a limited number of traits have been associated with markers in
A. hypogaea populations, including a RAPD marker linked to Diabrotica
undecimpunctata howardi Barber (corn rootworm) resistance and other RAPD
markers were associated with components of C. arachidicola resistance and with
plant color (Stalker and Mozingo 2001); AFLP markers have been associated
with resistance to aphids (Aphis craccivora Koch) by Herselman et al. (2004),

CAP markers with the high oleic acid content in oils (Lopez and Burow 2004), a
SSR marker with S. minor resistance by Chenault and Maas (2006), and a
RTPCR marker with drought resistance by Jain et al. (2001). Obviously, the
numbers of associations of molecular markers with peanut traits within A.
hypogaea is small and of limited value for breeding programs that select for
multiple resistances and quality characters.
In contrast to the A. hypogaea gene pool, molecular markers are highly
variable among Arachis species for many marker technologies, including iso-
zymes (Lu and Pickersgill 1993; Stalker et al. 1994), RFLPs (Kochert et al. 1991;
Paik-Ro et al. 1992), RAPDs (Halward et al. 1992), AFLPs (Gimenes et al.
2002; Milla et al. 2005a) and SSRs (He and Prakash 1997). Further, intraspe-
cific variation was found within the diploid species A. duranensis (Stalker et al.
1995) and selected markers are useful for identifying individual accessions.
Progenies of interspecific hybrids have been used to associate several traits
with molecular markers, including RFLPs with M. arenaria resistance (Burow
et al. 1996; Garcia et al. 1996) and components of C. arachidicola resistance
(Stalker and Mozingo 2001); RAPD markers with Empoasca fabae Harris
(leafhopper) and Cylindrocladium black rot resistances (Stalker and Mozingo
2001); and AFLPs with aflatoxin contamination (Milla et al. 2005b) and TSWV
resistance (Milla et al. 2004). In addition to markers being useful for associating
with specific traits, they hold promise for following gene introgression from
Arachis species to A. hypogaea.
9.8.2 Molecular Maps of Peanut
High-density molecular mapping in domesticated peanut has thus far not been
possible because of the low levels of molecular marker variation and the poly-
ploid nature of the species with homeologous chromosome pairs. To circum-
vent this problem, hybrid derivatives of species in section Arachis have been
utilized by several investigators. The first molecular map in peanut was created
by using RFLPs to analyze progenies of the cross A. stenosperma A. carde-
nasii (both A-genome species) (Halward et al. 1993). They mapped 117 RFLP
markers into 11 linkage groups. Garcia et al. (2005) developed a RAPD-based
linkage map of peanut based on a backcross population [A. stenosperma (A.
stenosperma A. cardenasii)] where 167 RAPD loci and 39 RFLPs also mapped
to 11 linkage groups; all common markers mapped into the same linkage groups
and mostly in the same order as in the Halward et al. (1993) map. A second map
was created by Burow et al. (1996) who grouped 383 RFLP markers into
linkage groups by analyzing progenies of a cross between A. hypogaea and
TxAG-6. They also associated the marker R239 with nematode resistance,
which mapped to the same linkage group in the map produced by Halward
et al. (1993). Unfortunately, both RFLP maps have low saturation levels and the
information does not directly translate into the polyploid species A. hypogaea.
9 Peanut 305
A microsatellite map was produced by Moretzsohn et al. (2005) with pro-

genies of the cross A. duranensis A. stenosperma (both A-genome species) that
clustered markers into 11 linkages groups. A second map with the B-genome
species A. ipaensis and A. magna Krapov., W.C. Gregory, and C.E. Simpson
was generated by utilizing the same SSR markers, and a comparison between
the A- and B-genome maps indicated that they were generally collinear (Gobbi
et al. 2006). Herselman et al. (2004) mapped 12 AFLP markers into five linkage
groups by using A. hypogaea crosses.
Creating a comprehensive physical map for peanut will require a very large-
scale effort with a library of large-insert DNA clones (Paterson et al. 2004).
Yüksel and Paterson (2005) produced a bacterial artificial clone (BAC) library
with 182,784 clones using the tetraploid peanut cultivar Florunner, and this
library should serve as a highly useful resource for developing a physical map.
However, it will be difficult to distinguish true homologes vs. homoeologs from
related species in BACs by hybridization-based molecular approaches because
of duplicate copies of genes from the two ancestral species (Paterson et al. 2004).
Lin et al. (2000) developed a method to determine the subgenome-specificity of
individual BAC clones in polyploid species, so an alternative method may be to
produce BAC libraries for diploid progenitors of A. hypogaea and then com-
pare libraries from the diploids and tetraploids (Paterson et al. 2004).
9.8.3 Gene Sequencing in Arachis

Arachis hypogaea has 5.91 pg DNA and A. duranensis ranges between 2.49 and
2.87 pg DNA (Temsch and Greilhuber 2000), with variation being observed at
different elevations and latitudes (Temsch and Greilhuber 2001). Singh et al.
(1996) concluded that the A and B genomes contributed nearly equal amounts
of DNA to the domesticated peanut. The large genome size and polyploid
nature of the domesticated peanut makes it an unlikely candidate to be com-
pletely sequenced. Further, the Arachis genome also has a high concentration of
repetitive DNA (Dhillon et al. 1980). An alternative to complete genome
sequencing is to develop libraries of expressed sequence tags (ESTs) which
can provide a significant amount of information about gene function. As of
June 2007, 12,832 long-sequence ESTs were deposited in GenBank, with the
majority of sequences from seeds. In addition, a large number of ESTs have
been produced from A. hypogaea seeds and leaves that are yet to be deposited in
GenBank (Guo et al. 2004; Chen et al. 2006; Stalker and Nielson, unpublished
data); and more than 25,000 unigenes have been identified from existing EST
datasets (S. Knapp 2007, personal communication). In addition to the long-
read ESTs, Jayashree et al. (2005) identified 1,312 short-read sequences that
were isolated from SSR-enriched libraries and S. Knapp (personal communica-
tion) is currently sequencing large numbers of short DNA segments with
methylation-filtration techniques.
Several genes found in peanut have been sequenced, including the D12-fatty
acid desaturase gene by Lopez et al. (2000), and several Ara h genes (which give
rise to proteins causing allergens in humans) by Viquez et al. (2003, 2004). Also,
homeologous Ara h 6 genes are present in diploid progenitor species, one in the
A-genome and two in the B-genome. Genomic sequencing and microarray-
based screening has been used to identify putative genes that may be associated
with resistance to Aspergillus parasiticus Speare and drought stress (Luo et al.
2005) and for aflatoxin contamination (Guo et al. 2003). In addition, Yüksel et
al. (2005) evaluated bacterial artificial clones from the BAC library and found
250 putative resistance gene loci in peanut.
9.8.4 Reverse Genetic Technologies

Gene discovery from Targeting Induced Local Lesions IN Genomes (TIL-
LING) populations is a method developed to find genes of interest in a mutant
population of a species. Because allergens in peanut cause a significant health
problem in the human population, a high priority of the peanut industry is to
eliminate (or suppress) the proteins that cause allergens. There are multiple seed
storage proteins that give rise to allergens, with the major ones being Ara h 1,
Ara h 2, and Ara h 3 (Burks et al. 1998); Ara h 2 is the most important for
causing human allergies (Koppelman et al. 2004). A TILLING population in
peanut is being created with the goal of eliminating Ara h 2 and possibly other
allergen genes (Ozias-Akins et al. 2006).
9.8.5 Peanut Transformation
Ozias-Akins et al. (1993) reported the first successful transformation and

accompanying plant regeneration in peanut by utilizing the micro-bombard-
ment technique. Micro-bombardment has since been completed in peanut with
a number of genes conferring disease resistance (Magbanua et al. 2000; Ozias-
Akins and Gill 2001; Higgins et al. 2004; Yang et al. 2004; Livingstone et al.
2003; Dar et al. 2006). Unfortunately, efficiency levels are low and the process
takes many months before plants mature (Egnin et al. 1998), and a high-
efficiency, rapid technique to transform peanut is greatly needed.
Chen et al. (1996) used an Agrobacterium-mediated transformation system
with a Valencia-type peanut. Sharma and Anjaiah (2000) published different
methodologies to transform peanut with Agrobacterium tumefaciens which
apparently works with a wider range of genotypes. Bhatnagar-Mathur et al.
(2007) utilized this methodology to develop transgenic lines with increased
transpiration efficiency to overcome drought stresses. To date, genetically
modified (GM) peanuts have not been sold in the marketplace. Before this
occurs, transgenic peanuts must be approved by governmental agencies [US
9 Peanut 307
Department of Agriculture’s Animal and Plant Health Inspection Service

(APHIS), the Food and Drug Administration (FDA), and the Environmental
Protection Agency (EPA)] and licensing agreements will need to be obtained for
the incorporated traits.
9.9 Seed Production
In the US, production of peanut seed follows the traditional seed certification
chain: breeder, foundation, registered, and certified. Specific purity standards
are required to meet certification. These include the time since peanut was
grown in the field, the distance from other peanut cultivars within the same
field, the percentage of contamination with other cultivars, crop or weed seeds
and in some cases, rules about seed storage and conditioning. Other than seed
saved by farmers under the US. Plant Variety Protection Act (PVP), nearly all
seed sold to farmers is certified by a state seed certification agency. The vast
majority of cultivars are protected by PVP which specifies that seed can be sold
only as a class of certified seed.
Producing high quality peanut seed requires somewhat more effort than
producing eatable peanuts. Most peanut seed are grown with irrigation and
optimum inputs of pesticides and fertilizers. One unique aspect of producing
high quality peanut seed is the fact that calcium content in the soil is critical to
producing seed with good germination. Most seed producers apply 625–900 kg/
ha of gypsum (calcium sulfate) when producing peanut for seed. Adams et al.
(1993) showed that calcium content of runner seed is vital for optimum germina-
tion. The amount of calcium to be applied varies depending on soil tests and the
cultivar being grown. Research demonstrates that cultivars with smaller seed
require less calcium than cultivars with larger seed, but there is also substantial
variation in calcium required by genotypes with similar seed size (Cox et al. 1982;
Walker et al. 1976). In fact, germination of some runner market-type cultivars
may not respond to gypsum application (Gomillion et al. 2007).
Most seed is grown by farmers under contract with peanut shelling compa-
nies. Each year, about 10% of the peanut acreage is devoted to seed production.
This may seem excessive, but as described in Section 9.7, peanut seeds are large
and the seeding density is relatively high. Peanut seed is about 10% of the
variable cost of producing the crop.
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Chapter 10
Castor
Dick L. Auld, Mauricio D. Zanotto, Thomas McKeon, and John B. Morris
10.1 Introduction
Castor (Ricinus communis L.) has the potential to become the premier vegetable
oil crop for industrial oil production across the globe (Roetheli et al. 1990).
Castor is an ideal candidate for production of high value, industrial oil feed-
stocks because of the very high oil content (48–60%) of the seed, the extremely
high levels of potential oil production (500–1,000 l of oil/acre), and this plants
unique ability to produce oils with extremely high levels (80–90%) of ricinoleic
acid (Brigham 1993). Additionally, the high potential yield and unique fatty
acid composition of castor allows this oil to provide economically competitive
feedstocks needed for the production of premium quality biodiesel, short chain
aviation fuels, fuel lubrication additives, and very high value biopolymers
(Geller and Goodrum 2004; Goodrum and Geller 2005; Roetheli et al. 1990).
Because castor is not used for food and can be grown productively on marginal
lands this crop represents a unique opportunity to expand industrial vegetable
oil production on a global basis. Historically, commercial production of castor
has been limited by concerns about the toxins found in castor seed, unstable
global markets for the oil and the lack of efficient technologies to produce and
process the crop (Brigham 1993). Development of improved production and
genetic technologies will help ensure rural regions across the world can partici-
pate in the economic potential of this crop.
Most castor seed contains approximately 50% oil which is composed of
80–90% ricinoleic acid (12-hydroxyl-cis-9-octadecenoic acid) (Atsmon 1989).
This unique hydroxy fatty acid is used in a number of processes to create
unique chemicals and polymers. Ricinoleic acid can be used in several bio-
based fuels and industrial products. Pyrolysis of methyl ricinoleate generates
methyl 10-undecylenate which can be processed to make Nylon 11 and a seven
carbon product (heptaldehyde) that can be used as an octane enhancer for
combustion engine fuels. Both of these products are highly valued industrial
D.L. Auld (*)

Department of Plant and Soil Science, Texas Tech University, Lubbock, TX, USA
e-mail: dick.auld@ttu.edu

318 D.L. Auld et al.
chemicals. A large variety of other reactions have been described that produce
other high value products with great potential as biofuels and industrial
polymers.

Castor was probably one of the first crops cultivated by early man who used
the oil extracted from the seed for a wide variety of uses including lamp oil
(Weiss 2000). Castor is a member of the Euphorbiaceae family that is thought
to have originated in Eastern Africa but has spread through out the tropical
regions of the world. Castor is a diploid (x = 10, 2n = 20) with few if any
natural polyploids (Moshkin 1986). Early taxonomists tried to classify
castor (Ricinus communis L.) into several subspecies based on phenotypic
differences but most botanists now believe all castor belong in the same
species. Castor accessions display significant differences in height, branch-
ing, color or growth habit, and many of these phenotypic traits are simply
inherited (Peat 1928; Fig. 10.1) and most accessions will readily intercross
(Atsmon 1989).
Fig. 10.1 Phenotypic variation for capsule spines in castor (Ricinus communis L.) showing
spineless capsules (ss), reduced spines (Ss), and normal spines (SS; Peat 1928). Photos:
A.D. Limmer, Texas Tech University
Most castor accessions produce monoecious flowers with the male (stami-
nate) flowers on the base of the inflorescence and the female (pistillate) flowers
located on the terminal end of inflorescence (Atsmon 1989). However, the
relative proportion and the location on the inflorescence of the male and female
10 Castor 319
flowers vary between different genotypes. Castor is highly cross pollinated with
estimates on the High Plains of Texas ranging from 70 to 90% (Brigham 1967a).
Consequently, self pollinated seed can only be produced by sacking individual
inflorescences prior to flowering (Atsmon 1989).
Historical summaries of castor production have been published by both
Atsmon (1989) and Weiss (2000). In 2005 and 2006, India, China and Brazil
produced the majority of the world’s castor oil with Ethiopia, Thailand and
Paraguay contributing relatively minor amounts (Table 10.1). Annual total
world production of castor seed exceeded one million tons during this period
but average seed yields were never more than 1,200 kg/ha during this period. The
volatility of castor oil prices and variability in production has made the interna-
tional market for castor oil very unstable (Roetheli et al. 1990). Increasing
demand for vegetable oils for biodiesel and other industrial applications have
increased interest in improving the genetics and production of castor worldwide.
Table 10.1 2006 and 2007 average castor seed production area, seed yield and total seed
production in six major producing countries and world wide (FAO-STAT 2008)
Country Production area (ha) Seed yield (kg/ha) Total production (MT)
India 805,000 1,063 861,000
China 255,000 961 245,000
Brazil 184,231 701 130,565
Ethiopia 14,500 1,034 15,000
Thailand 13,430 781 10,492
Paraguay 10,000 1,100 11,000
World 1369,720 956 1314,193
10.3 Varietal Groups
Castor has not been divided into varietial groups in recent times but most
breeders recognize tall, late flowering types as tropical in origin. Those types
which flower and mature quickly are usually adapted to either high altitude
environments or more temperate latitudes in either the northern or southern
hemisphere. There have been no reported barriers to intercrossing the two types
and obtaining segregating populations.
A search of International Germplasm collections on the Bioversity web site

combined with the USDA-ARS castor germplasm at Griffin, GA (USA)
identified 12 major sources of germplasm and a total of 6,588 accessions
(Table 10.2). Extensive germplasm collections are held in Brazil, China, Ethio-
pia, India, Kenya and the former USSR, but availability of these germplasm
resources is not known. Additional castor germplasm can be obtained from
Table 10.2 Major germplasm collections of castor (Ricinus communis L.) as listed by the
Bioversity International Directory (October 14, 2008)
Country Collection agency Accessions reported
Brazil CENERGEN/EMBRAPA 360
Brazil Centro Nacional de Pesquisa de Algodao (CNPA) 199
Brazil Empresa Baiana de Desenvolvimento Agricola 528
S.A.
Brazil Instituto Agronomico de Campinas (I.A.C.) 200
China Institute of Crop Science (CAAS) 1,689
China Institute of Oil Crops Research (CAAS) l,652
Ethiopia Biodiversity Conservation and Research Institute 232
India Region Station Akola, National Bureau of Plant 290
Genetic Resources (NBPGR)
Kenya National Dryland Farming Research Station, 130
Kenya
Kenya National Genebank of Kenya, Crop Plant Genetic 43
Resources Centre, KARI
Romania Agricultural Research Station Teleorman 66
Russia N.I. Vavilov All-Russian Scientific Research 423
Institute of Plant Industry
Serbia Maize Research Institute 69
Serbia Institute of Field and Vegetable Crops 43
Ukraine Institute for Oil Crops 255
United USDA-ARS-PGRCU 364
States
World 39 Institutes 6,588
public breeders in South America including Brazil and Columbia. In tropical

climates world wide, castor can be found as an introduced plant species surviv-
ing as a weed in roadsides and non-cultivated areas. This feral castor can be
a valuable source of germplasm especially for adaptation to localized diseases,
pests and environmental conditions.
10.5.1 Fatty Acid Composition

In 2004, a natural mutant of castor was isolated from USDA Plant Introduction
PI 179.729 that had increased concentrations of oleic acid and reduced levels of
ricinoleic acid (Rojas-Barros et al. 2004). This mutant produced seed oils with
approximately 78% oleic acid and from 10.1 to 18.8% of ricinoleic acid. The
increased levels of oleic acid appeared to be controlled by two independent
genes (ol, Ml) with epistatic interaction (Rojas-Barros et al. 2005). Incorpora-
tion of this mutant and other high oleic acid mutants in improved varieties may
enhance the use of castor oil as a biodiesel feedstock.
10 Castor 321
Castor oil biosynthesis is a matter of considerable biochemical interest, due to

the unusual chemical nature of the ricinoleate hydroxylation – a stereospecific,
geometrically specific hydroxylation of a hydrocarbon chain, still an unrealized
dream for the synthetic organic chemist. Although the cloning of the gene for this
enzyme, oleoyl-12-hydroxylase, provided interesting insights into the production
of ricinoleate and other uncommon fatty acids, transgenic plants that expressed
the hydroxylase gene produced oils containing less than 20% hydroxy fatty acid
(Broun and Somerville 1997).
Since the hydroxylase gene alone was not sufficient to elicit high levels of
hydroxy fatty acid production, it seemed that there must be other enzymes
that are required in order to achieve high ricinoleate levels in oil. Based on
metabolomic studies of castor oil biosynthesis carried out by castor seed
microsomes, there are several enzymatic steps that appear to be important
for high ricinoleate levels (McKeon and Lin 2002). Further studies (Lin et al.
2002) indicated that 6-fold more ricinoleate is incorporated into triacylgly-
cerol (TG) than oleate. This result indicated the final step in oil biosynthesis,
diacylglycerol acyltransferase (DGAT), as the reaction that led to high
ricinoleate content and minimal oleate in the oil. The diacylglycerol acyl-
transferase (DGAT) is a transmembrane enzyme that catalyzes the acylation
of diacylglycerol (DG) to TG, using acylCoA as the source for the final acyl
group. The DGAT reaction is widely considered to be the limiting step in oil
biosynthesis, with considerable evidence indicating that altered DGAT activ-
ity levels dramatically affect the yield of oil (He et al. 2005). With the cloning
of the DGAT1 from developing castor seed, it has been demonstrated that
the activity and protein level of the cloned DGAT is closely correlated with
the onset of oil biosynthesis in the seed (He et al. 2004). The castor DGAT
enzyme displayed a 2-fold preference for using diricinolein vs. other non-
hydroxylated fatty acyl DG. The acyl-donor for the DGAT reaction is
produced by the acylCoA synthetase (ACS). One of several ACS cloned
from castor displays a threefold preference for condensing ricinoleate vs.
oleate with CoASH (He et al. 2007) suggesting the combined effect of the
enzymes from the two cloned genes could result in a significantly higher
incorporation of ricinoleate vs. oleate into oil. The question of how the
castor seed produces an oil with such a high proportion of ricinoleate and
a high oil content, approaching 60% continues to provide a challenge.
Understanding this process may ultimately lead to increased oil content
and the ability to engineer the production of other uncommon and indust-
rially useful fatty acids in the castor seed.
10.5.2 Castor Toxins

Ricin is a protein toxin found in the endosperm of mature castor seed that is
capable of inhibiting protein synthesis by enzymatically destroying the ribo-
somes of eukaryotes (Khvostova 1986). The presence of ricin in the high protein
meal of castor remaining after oil extraction has historically reduced its value as
an animal feed (Roetheli et al. 1990). Since ricin has the potential to be extracted
and used as a chemical warfare and bioterrorism agent, the production and
processing of castor has undergone increased scrutiny by international law
enforcement agencies since the terrorist attacks of September 11, 2001 (Lowery
et al. 2007; Franz and Jaax 1997). Development of castor cultivars with reduced
levels of ricin would improve the economics of castor oil production, reduce the
potential for accidental poisoning and eliminate the potential of ricin being used
by terrorists.
Ricin has both an A and a B chain linked together by a disulfide bond. Both
the A and B proteins have been DNA sequenced and appear to be initially
produced by a single gene in castor (Halling et al. 1985; Tregear and Roberts
1992). Ribosome-inactivating proteins such as the ricin A chain typically contain
a N-glycosylated, 32 kDa monomer (Olsnes and Pihl 1973). The A chain is
attached to the cell surface by a the 34 kDa protein (B chain) (Roberts et al.
1985; Frankel et al. 1989). The ricin A chain has also been used in the production
of immunotoxins which target specific diseases in humans (Ghetie and Vitetta
1994). Castor meal when applied as an amendment to greenhouse potting media
has been shown to improve growth of okra (Hibiscus esculentus) and suppress
root-knot nematode (Meloidogyne arenaria) (Ritzinger and McSorley 1998).
Ricin has historically been degraded by exposure to high temperature for two
or more hours (Roetheli et al. 1990). Ricin can also be degraded by exposure to
concentrations of 3 mM sodium hypochlorite (Mackinnon and Alderton 2000).
Castor seeds also contain a second toxin, Ricinus communis agglutinin
(RCA120) (Hartley and Lord 2004). RCA120 is very similar in both amino
acid sequence and structure to ricin (Hartley and Lord 2004). RCA120 is
composed of two A-chains and two B-chains linked together by disulfide
bonds. This compound is much less toxic to mammals than ricin (Lowery
et al. 2007).
Ricin makes an ideal target for genetic manipulation since this toxin appears
to be controlled by a single gene that encodes both the A and B chains of castor
(Halling et al. 1985). Conventional genetics were used to reduce the levels of ricin
in dwarf-internode castor using crosses with two accessions from the Soviet
Union, PI 258 368 and PI 257 654, which had been previously selected for reduced
levels of ricin (Khvostova 1986). In subsequent segregating generations, indivi-
dual plants were selected for dwarf-internode growth habit and reduced levels of
ricin and RCA120 using a radial immunodiffusion (RID) assay (Auld et al. 2003;
Auld et al. 2001; Pinkerton et al. 1999). In 2003, twelve F8 lines where intercrossed
to develop a synthetic population adapted to mechanical harvest. In 2004 and
2005, this population was screened for semi-dwarf internode growth habit and
lack of shattering. This process developed a new experimental castor variety,
‘Brigham’ which has a ten fold reduction in the level of ricin.
A third toxic substance found primarily in the capsules and seed hulls of
castor is the alkaloid, ricinine (Bukhatchenko 1986). Ricinine is thought to be
product of specific nitrogen synthesis and consists of a monocyclic derivative of
10 Castor 323
pyridine which carries a cyanide group. It appears to be a naturally occurring

insecticide in castor that has a relatively low human toxicity. Russian research-
ers reported a negative correlation between the concentration of ricinine and oil
in castor seeds. It also appears that drought conditions during seed maturation
enhance the concentration of ricinine.
10.5.3 Castor Allergens

The allergy caused by exposure to the plant tissue and residue of castor has
historically caused human health problems to those individuals working
around or in close proximity to castor fields or plants processing castor seed
across the globe (Panzani and Layton 1963). Most of those individuals expres-
sing an allergic reaction have asthma like symptoms (Mercier and Panzani
1988). Cross-reactivity can occur with other species of plants within Euphorbia-
ceae (Layton et al. 1970). There has not been sufficient research to describe the
chemicals produced by castor that cause these allergic reactions or germplasm
screening to see if castor genotypes differ in their relative ability to cause allergic
reactions in humans. However, the development of castor cultivars which
do not cause allergic reactions would enhance production and processing of
this crop.
10.5.4 Qualitative Traits
Capsule drop resistance caused by pathogens such as Botryotinia ricini

(Godfrey) Whet. in humid areas of the USA is controlled by one or possibly
two genes (Culp 1966). At least one of these genes appeared to be closely linked
to the short pedicel trait. Researchers in both Russia and Trinidad have con-
ducted exhaustive genetic studies that show the color of stems, leaves and
capsules (Fig. 10.2), waxy coat on the stem and petiole, color of the hypocotyl
and stigma of flowers, spines of the capsules, dehiscense of capsules, pedicel
length, color and form of the seeds (Fig. 10.3), female sterility, flowering period,
seed hull color, plant height and numerous other phenotypic traits are fairly
simply inherited (Moshkin 1986; Peat 1928).
10.5.5 Quantitative Traits
In castor as well as in the majority of cultivated plants, the agronomic char-

acteristics of primary economic importance are inherited in a quantitative
manner including seed and total biomass production.
Fig. 10.2 Phenotypic variation in pod color of castor (Ricinus communis L.) caused by
different combinations of the M (Mahogany) and G (Green) genes (Peat 1928). Photos:
A.D. Limmer, Texas Tech University
10 Castor 325
Fig. 10.3 Phenotypic variation in seed shape, size and color of several different accessions of
castor (Ricinus communis L.). Photo: A.D. Limmer, Texas Tech University
Hooks et al. (1971) evaluated the behavior of inbred lines of castor in a diallele
cross using the method of Gardner and Eberhart (1966) and the procedures of
Hayman (1954 and 1958). They observed that additive genetic effects were
important in the initiation of bloom, the number of racemes per plant, and seed
oil content. However, the number of nodes prior to flowering showed significance
of additive genetic effects which agreed to the estimates derived by Swarnlata
et al. (1984). Giriraj et al. (1974) evaluated a diallele cross of six geographically
diverse cultivars and evaluated the length of the primary raceme, the number of
capsules per primary raceme and 100-seed-weight. They showed that these traits
also had very significant additive genetic effects. Solanki et al. (2003) evaluated
the genetic effects of eight agronomic characteristics with similar results.
Solanki and Joshi (2000) evaluated a diallele cross between monoecious and
female plants and found that additive effects were primarily responsible for the
number of nodes, the length of the primary raceme, number of racemes per
plant, number of capsules per primary raceme, 100-seed-weight, and total
weight of seeds produced 120 and 240 days after the sowing. This study also
showed that the characteristics of days to 50% of bloom of the main cluster,
100-seed-weight and height of plants had high heritabilities indicating rapid
selection efficiency.
Russian researchers have conducted extensive investigations on the inheri-
tance of the components of seed yield and oil content in castor with similar
results (Moshkin 1986).
Castor is an often cross pollinated species with 14–45% self pollination under
tropical conditions. In castor, the procedures for making artificial crossings and
self-fertilizations are relatively easy and result in several seeds. Because of the
reproductive biology of this species, the methods used to improve self pollinated
plants as well as the process of recurrent selection that is most often used on
cross pollinated crops are feasible.
10.6.1 Mass Selection
Mass selection is most effective for characteristics with high heritabilities in

populations with high levels of natural genetic variability such as heterogeneous
land races. Two procedures are useful in increasing the efficiency of the mass
selection in populations of castor: The self-fertilization of the selected plants to
prevent cross pollination, and the use of controlled selection techniques to
reduce environmental variation. Moshkin (1967) cites examples of application
of the mass selection for enhancement of female flowers, in the selection for
resistance to Fusarium, and reduction of plant height. Savy Filho (2005) used
mass selection to develop IAC-38, an important dwarf castor cultivar in Brazil.
10.6.2 Individual Plant Selection with Progeny Tests

As in the mass selection, the selection of individual plants based on progeny
tests is highly effective for the improvement of populations of castor with
high levels of natural genetic variability. In the case of simultaneous selec-
tion for several characteristics, selection for the characteristics must be made
using the highest possible number of self pollinated lines and be preceded
first by the traits with high heritability that can be identified without the use
of replicated trials. Subsequent selection and the final evaluation should be
done on about 200 inbred lines arranged in a lattice experimental design
with 3–4 replications in two or three locations and years. This type of
testing allows selection for high seed and oil yield. In all phases of selection,
the use of appropriate commercial check varieties will enhance selection
10 Castor 327
efficiency. Amaral (2003) successfully used the individual plant selection

followed by progeny tests in developing the cultivar ‘Guarany’ of castor
with increased seed yield.
10.6.3 Methods Involving Sexual Hybridization

When populations of castor with sufficient natural genetic variation for
agronomic characteristics are not available, it is necessary to produce sexual
hybrids between different lines or cultivars to generate sufficient genetic
variability to support a selection program. The choice of the parents of these
populations must be based on their agronomic performance within the
targeted production region. In the case where there are several promising
parents or cultivars it may be necessary to use a diallele cross design to identify
those potential crosses with the greatest potential for creating highly perform-
ing sexual hybrid populations.
10.6.3.1 Pedigree Method

In the F2, F3, and F4 generations, self pollinated individual plants are selected to
derive uniform lines. During this process, selection is practiced both between
lines and within lines by selecting only the best plants within the best lines. By
the F5 or F6 generation the inbred lines should have a high degree of homo-
zygosity. These lines are then subject to a final evaluation using an experimental
design and statistical interpretation of data taken over multiple locations and
production years. Those inbred lines with superior performance that out-yield
the check cultivars can be increased to create a new commercial cultivar.
The pedigree method works best when it is necessary to select simultaneously
for several characteristics. Selection for the characteristics with the highest
heritabilities should be made in the initial generations (F3 and F4), and the
selection of quantitative characteristics in later generations. The pedigree
method is limited by the selection of initial plants in the F2 generation needed
to produce F3 inbred lines. This early generation selection process can restrict
the full expression of individual plants genetic variability since F2 plants are
highly heterozygous.
10.6.3.2 Bulk Method

The bulk method allows the hybrid population to segregate without artificial
selection until the F5 or later generations (F6 or F7). The bulk method is most
effective when the main objective of the program is to improve the adaptation of
castor to stress conditions such as drought, acid soils, high levels of salt and
resistance to diseases. After selection elite lines undergo a final evaluation as
described for the pedigree method.
10.6.3.3 Single Seed Descent Method

The use of the single seed descent (SSD) method in the improvement of castor
has not been frequently practiced but it offers two interesting aspects. It does
not allow either natural nor artificial selection during the segregating genera-
tions but it does allows the increase of up to two generations per year using off
season increases in either winter nurseries or greenhouses. In addition this
techniques does not have as drastic reduction in the genetic variability in the
F2 or F3 generations as the pedigree method.
10.6.3.4 Backcross Method

The backcross method of selection is most effective when there is a need to
improve some simply inherited, qualitative characteristic in a commercial culti-
var or promising elite line. The non-recurrent parent must have the character-
istic absent from the recurrent parent. The method of backcrossing is especially
effective in castor for the improvement of characteristics such as seed shatter-
ing, flower height, and disease resistance.
10.6.3.5 Recurrent Selection

Recurrent selection can be defined as successive cycles of selection and recom-
bination of selected lines or individual plants. Recurrent selection has been
more extensively used in species such as maize rather than in castor; Zanotto
et al. (2004) had considered recurrent selection with use of inbred lines for the
reduction of plant height in the cultivar ‘Guarani’. The selection cycles occurred
in two stages. In the first stage short plants were selected and self pollinated
from the cultivar ‘Guarani’. In the second stage, 180 self pollinated lines were
evaluated for plant height in isolation and racemes of five plants of each one of
the 30 selected lines were self pollinated by paper bags as described by Savy
Filho (1999). After the selected lines had gone through at least five cycles of self
pollination, the 30 selected lines were intercrossed and the harvested seed mixed
to generate the cycle 1 seed. This procedure was repeated for four additional
cycles of selection. According to Oliveira and Zanotto (2008) plant height was
reduced by 28 cm, 13 cm, 19.9 cm, 11.7 cm and 3.4 cm, respectively, for the five
cycles of selection. This process demonstrated the effectiveness of using recur-
rent selection with self pollination in castor.
Allergen and ricin content are major issues that affect interest in production and
processing of castor seed for castor oil. Since castor is not a food crop, one
potentially fruitful approach to eliminating these noxious proteins is genetic
engineering, to silence their expression during seed development. Both the
primary allergen, 2S albumin, and ricin are expressed at a very high level during
10 Castor 329
seed development (Chen et al. 2004; Chen and McKeon 2005). With the proper
choice of promoter and application of gene silencing techniques, gene expres-
sion can be suppressed up to 10,000 fold, which would be adequate for safe
exposure to the seed meal.
However, genetic transformation of castor has proven to be highly challen-
ging, as it is recalcitrant to efficient regeneration of stably transformed plants.
The first report of transformed castor (McKeon and Chen 2003) described a
vacuum infiltration technique using Agrobacterium carrying marker genes.
Since then, an apparently more efficient method has been developed (Sujatha
and Sailaja 2005). However, given the need to reduce ricin content by a factor of
10,000 and a similar reduction of allergen, there remains an urgent need to
improve the efficiency of castor transformation. With such a method, not only
could the noxious proteins be virtually eliminated, improvements that would
benefit the growth and production of castor such as herbicide resistance, pest
resistance, or monoracemic fruit-bearing could be made available. These traits
would enhance the productivity of castor and simplify its harvesting. As a
monospecific genus, Ricinus communis is somewhat limited in germplasm avail-
ability. The ability to introduce foreign genes into this non-food crop would
have great potential to expand its growth habit and productivity.
10.8 Seed Production
Heterosis was shown to have a significant impact on days to flowering, racemes

per plant, volume weight, oil content and seed yield indicating that increased
seed and oil yields could be expected from the production of hybrids of castor
(Hooks et al. 1971). Hybrids also appeared to have faster seedling emergence
than open-pollinated lines (Brigham 1965). Composites have been used to
capture a portion of this heterosis and increase genetic variability in castor
(Brigham 1973). The production of hybrid seed in castor has been achieved
using the f N-pistillate gene for female racemes and environmentally sensitive
genes of interspersed-staminate flower by producing seed in locations with
cooler temperatures (75–838F) (Zimmerman and Smith 1966). In addition,
two female-sterile characters (fs1 and fs2) were identified by Brigham (Brigham
1967b). Commercial hybrids are now used in commercial production in Brazil,
India and other parts of the world.
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Chapter 11
Oil Palm
Aik Chin Soh, Choo Kien Wong, Yuk Wah Ho, and Chieh Wean Choong
11.1 Introduction
The oil palm is the world’s most important oil crop producing 24.9% of total
vegetable oils and fats surpassing soybean at 23.9% (Mielke, http://www.oil
world.bz, 31 March 2007). It produces two types of oil from its fruits, meso-
carp oil and kernel oil known as crude palm oil (CPO) and palm kernel oil
(PKO), respectively, in international trade. Total world production of CPO
stands at about 38 million tons worth around US$ 20 billion. The oils are
produced from some 13 million ha of plantations in the humid tropical
countries of Asia, Africa and Latin America: Indonesia (5.3 million ha),
Malaysia (4.2 million ha), Papua New Guinea, Colombia, Ivory Coast,
Nigeria and Thailand with the first two countries having the bulk of the
plantings. Palm oil is the largest internationally traded vegetable oil with its
main markets in China, European Union, Pakistan, India, Japan and Bangla-
desh. Palm oil is mainly used in food (80%), e.g. as cooking oil, margarine,
vanaspati or vegetable ghee and shortenings, and the remaining 20% are used
as oleochemicals replacing mineral oil to feed the detergents, cosmetics,
pharmaceutical/nutraceutical, plastics and lubricants industries. With the
recent high rise in petroleum prices and that the deadline for meeting the
requirements of the Cartagena Protocol on Biosafety in terms of ‘green’ or
renewable energy substitution is approaching, there has been a tremendous
demand for palm oil as a source of biofuel (biodiesel). Also, responding to
consumer health and environmental concerns, secondary and by-products
from the palm oil industry have spawned new industries, e.g. vitamins A and
E and other antioxidant health supplements from the oil, animal feed and
organic fertilizers from the kernel, and sludge cakes and wastes from oil
extraction mills have served as value additions.
A.C. Soh (*)

Applied Agricultural Resources, Locked Bag 212, Sg. Buloh, Post Office, 47000 Sg
Buloh, Selangor, Malaysia
e-mail: sohac28@gmail.com

334 A.C. Soh et al.
The oil palm has been postulated to originate in Gondwanaland which disap-
peared when the American and African continents drifted apart in prehistoric
times (Zeven 1965) giving rise to the evolution of African oil palm (Elaeis
guineensis Jacq.) and American oil palm (E. oleifera or E. melanoccoca pre-
viously). The African oil palm is endemic to the equatorial belt of Africa
stretching eastwards from Guinea at the Atlantic coast to Madagascar Island
in the Indian Ocean and from Senegal in the sub-Sahara to the Angola-Namibia
border in the south. However, the centres of origin and diversity appear to be
concentrated in the tropical forests of the west (Ivory Coast, Nigeria, Ghana,
Cameroon) and central (Congo, Zaire, Angola) African countries where they
occur as semi-wild forest groves fringing rivers in the lowlands usually close to
settlements, although they can thrive in drier and higher areas (Hartley 1988;
Latiff 2000). The American counterpart is endemic to the tropical countries of
Central and South America, from Mexico in the north to the Amazon in the
south and from the Pacific to the Atlantic coasts. Its distribution appeared to be
more discontinuous, it is also found in open grasslands and river banks and
associated with native Indian migratory trails (Santos et al. 1986). The oil palm
fruit forms an important part of both the native West African and South
American diet as a source of fat, carbohydrate, protein and vitamins.
Interest in oil palm as an industrial crop arose from the need to substitute
animal fat in the production of candle wax, soap and margarine. The European
colonists started oil palm plantations in Indonesia and Malaysia to ensure a
steady supply of oil. Four seedlings planted in Bogor Botanic Gardens in
Indonesia derived apparently from the same fruit bunch in West Africa and
obtained via Amsterdam and Mauritius/Reunion, gave rise to the current
industry. Hybridizations and selections among the progenies of these four
progenitors, which had thick shell fruits or Dura (D) fruit form, were distrib-
uted to the plantations in Deli province in Sumatra and thence to Malaysia
(Rosenquist 1986). These Deli Ds were the commercial planting material for the
rapidly developing plantation industry in Malaysia and Indonesia from 1911 till
the early 1960s. Beirnart and Vanderweyen (1941) elucidated the monogenic
inheritance of the shell gene: the cross between the thick shell D parent palm and
the shell-less (usually female sterile) pisifera (P) parent would give rise to 100%
thin-shell tenera (T) palms (Fig. 11.1) exhibiting incomplete dominance of the
shell gene; the T T, the T P and D T crosses would give rise to segregating
progenies in the classical Mendelian ratios of 1D:2T:1P for the first (Fig. 11.2),
1T:1P for the second and 1D:1T for the last cross, respectively. Teneras from
West Africa have been brought in by breeders and with this revelation the
switch to the T hybrid as the commercial material was very rapid (Hartley
1988). This process was spurred on by private plantation companies becoming
interested in commercial hybrid seed production and investing in oil palm
breeding. Mixed T or D P hybrids have been the dominant commercial
11 Oil Palm 335
Fig. 11.1 Commercial dura

pisifera hybrid palm seed
production
Fig. 11.2 Monogenic inheritance of shell thickness: F2-segregation
planting materials till today. Recently, oil palm clones from tissue culture have
become commercially available although still in limited quantities as compared
to the total demand for oil palm seeds (Soh et al. 2006).
In oil palm, there are no varieties in the strict sense as the commercial materials
are mixed hybrids from non-fully inbred and sometimes out-crossed parents
(Soh 1999). They can therefore be considered as inter-population hybrids. The
parent populations e.g. Deli, were usually derived from very few progenitors
336 A.C. Soh et al.
and have being accordingly referred as BPROs (breeding populations of

restricted origins) by Rosenquist (1986). Only a handful of BPROs are featured
in commercial hybrid breeding programmes:
Deli. This BPRO is featured almost exclusively as the female parent in all
commercial hybrid seed production programmes. The distribution of this
BPRO to various countries followed by local selection led to the development
of a number of subpopulations, e.g. Ulu Remis Deli, Elmina Deli, Serdang Deli
(Malaysia), La Me Deli, Dabou Deli (Ivory Coast). The Dumpy and Gunung
Malayu are dwarf mutants of the Deli.
AVROS. The ancestral palm was the ‘Djongo’ or ‘Best’ palm found at Zaire’s
Eala Botanical Garden. Seeds were planted in Sungai Pancur, Sumatra by
AVROS (Algemeene Vereniging van Rubberplantera ter Oostkust van Suma-
tra) in 1923 giving rise to the well-known SP540T parent. Subsequent crosses
with other T palms in Bangun Bandar Experimental Station were followed by
backcrosses to SP540T selfs and became the AVROS BPRO. AVROS seeds
were brought into Malaysia and their subsequent progenies distributed widely
in Malaysia, Indonesia and thence to Papua New Guinea, Colombia, Costa
Rica and Thailand. The major oil breeding and seed production programmes in
these countries are based on the AVROS Ps or its derivatives. Vigorous trunk
growth and high oil yield from big fruits with thick mesocarp are characteristic
features of this BPRO. Pure descendents of SP540T gave rise to the RISPA
BPRO in Indonesia.
Yangambi. The breeding programme of INEAC (Institut National pour
l’Étude Agronomique du Congo) at Yangambi station involving open-
pollinated progenies of the Djongo palm and other Ts from Yawenda,
Isangi and N’gazi gave rise to this BPRO. This lineage is also featured in a
number of major breeding programs as a source of Ps in D P hybrid
seed production. Yangambi BPRO shares similar features of vigorous palm
growth, high oil yield from big fruits with thick mesocarp as the AVROS.
A short variant (16R) was found and developed in this population.
La Me. This BPRO was bred by IRHO (Institut de Recherches pour les
Huiles et Oleagineux) currently known as CIRAD (Centre International
Recherche Agricola et Développement) from the 21 T palms, particularly
the Bret 10 palm, derived from seeds collected from wild groves in Ivory
Coast. This BPRO, characterized by its smaller palm stature, production of
many smaller bunches with small fruits and generally tolerant of less
favourable growing conditions, forms the genetic base of the breeding and
seed production programmes in West Africa and Indonesia advised by
CIRAD.
Ekona. Using wild palms found in the Ekona area of Cameroon, breeding by
the Unilever plantations group at the Cowan, Ndian and Lobe estates led to the
development of this BPRO. Largely through the Unilever plantations group
network, the Ekona BPRO, particularly progenies from the Fusarium wilt
resistant and high oil yielding palm, CAM 2/2311, were distributed to Malaysia,
11 Oil Palm 337
Costa Rica and Thailand. This BPRO confers smaller fruits but with high oil
content to their progenies.
Calabar. The genetic base of the Nigerian Institute for Oil Palm Research is
much broader from collections made in Aba, Calabar, Ufuma and Umuaibi.
Progenies from its Calabar selections, particularly palm NF 32.3005 have been
distributed to Ghana, Costa Rica, Indonesia and Malaysia.
Derived and recombinant BPROs. A number programmes have initiated the
recombinant phase of breeding having interbred or introgressed parents from
various traditional BPROs to form new BPROs with mixed lineages or recom-
binant BPROs, e.g. URT (Ulu Remis teneras), Dumpy.AVROS, Dumpy.
Yangambi.AVROS, La Me x Yangambi, La Me x Dumpy.AVROS (Soh
et al. 2006).
Recognizing the very narrow genetic base (Deli, AVROS, Yangambi, La Me)
of the existing oil palm breeding programmes and considering that Malaysia
had a very high stake in the rapidly expanding industry, MARDI (Malaysian
Agricultural Research and Development Institute) and subsequently PORIM
(Palm Oil Research Institute of Malaysia), currently known as MPOB
(Malaysian Palm Oil Board), launched a series of expeditions to Africa and
Latin America to prospect for new E. guineensis and E. oleifera germplasm for
genetic base broadening, breeding and conservation in its ex situ genebank
(Ooi et al. 1973; Rajanaidu et al. 1979; Obasola et al. 1983; Rajanaidu and
Rao 1988). The first prospection was systematically done in Nigeria in colla-
boration with NIFOR and the then International Board of Genetic Resources
with the objectives of genetic conservation and studying its population genetic
structure. Genetic studies done on the prospected progenies revealed that
genetic variability was higher within families than between families and popu-
lations. Subsequent collections in Angola, Cameroon, Gambia, Guinea,
Madagascar, Sierra Leone, Tanzania and Zaire were guided by this finding
and also targeted towards accessions with prospective economic and agro-
nomic traits. Prospections for E. oleifera in Latin America were carried out in
Brazil, Colombia, Costa Rica, Ecuador, Honduras, Panama, Peru and Sur-
iname and included also other oil bearing palms (Bactris gossipaes or Pejibaye,
Jessenia-Oenocarpus, Orbignya martiana or Babassu) with unusual fatty acids
and other uses. The accessions have been planted and maintained as living
collections in Malaysia with a sample retained by the host country (Rajanaidu
1990; Rajanaidu and Jalani 1994). These prospections proved to be scientifi-
cally successful in that a number of extremely desirable traits and traits absent
in existing breeding populations have been discovered (Table 11.1) and intro-
gressed into advanced breeding populations or developed into new popula-
tions (Sharma 1999; Rajanaidu et al. 2000).
338 A.C. Soh et al.
Table 11.1 Oil palm collections from various countries and their special attributes
Accessions Useful traits
A. E. guineensis
Nigeria High in unsaturated fatty acids, dwarfness, high stearic acid, low lipase
Angola Large fruits, high carotene content
Zaire Tolerance to Ganoderma disease
Cameroon Tolerance to Ganoderma disease
Tanzania Thin shell
B. E. oleifera
Colombia Very high unsaturated fatty acids content
Brazil Thicker oil bearing mesocarp
Costa Rica Compact stature, good oil yielding
Ecuador Thicker oil bearing mesocarp, very small palms
Suriname Very compact palm tree stature

11.5.1 Tenera Hybrid Improvement
Davidson (1993) attributed 70% of the oil palm yield improvement in Malaysia
for the previous 50 years to breeding improvement and 30% to improved
agronomic practices. Undeniably, the mere switch-over from the thick shell,
thinner oil-bearing mesocarp (ca. 60% mesocarp to fruit content) D to the thin
shell, thicker mesocarp T (ca. 80%) variety would account for at least 30% of
the yield increase, notwithstanding improved FFB yield. There have been at
least two generations (ca. 20 years) of improved T materials since and oil yields
have improved from about 5 t/ha/yr to about 10 t/ha/yr based on trial results
then. Hardon et al. (1987) estimated that there had been an average yield
improvement of about 15% per generation over two generations of breeding
in the D, but they did not translate this into T hybrid improvement. Subse-
quently, Lee et al. (1990) and Rajanaidu et al. (1990) estimated a resultant T
improvement of only 6–7% when they progeny tested the same Ps on two
successive generations of selected Ds. Lee and Yeow (1985) reported that
selecting the best P (top 15%) from the progeny-test of seven Ps would give
12% improvement. In commercial seed production, at least 3–4 progeny tested
Ps (top 30–50% selection) need to be used reducing the improvement to about
5%. Reconciling the estimated improvements in the two parental populations,
the estimated improvement of 10–15% per generation for the T hybrids was not
unreasonable (Soh et al. 2003a). The grossness of the estimate is inevitable as
there were no common standard crosses linking the different trials of different
generation materials and the difficulty of identifying a standard (single hybrid,
sample of mixed hybrids) treatment to represent a particular generation of
commercial mixed hybrids. Breeding programmes based on the MRRS (mod-
ified reciprocal recurrent selection) system (Soh et al. 1999) were better placed
11 Oil Palm 339
to compute breeding progress more objectively. The original progeny tested

hybrid selected for reproduction as commercial hybrid (using the selfs of the
parents) was used consistently as the standard cross. Likewise, the superior
hybrid selected for reproduction as the second cycle commercial hybrid can be
used as a standard for comparing subsequent cycle hybrids. Oil yield improve-
ments of 18% (Gascon et al. 1988) and 25% (Lubis et al. 1990) in the first cycle
of MRRS, and 10–15% in the second cycle (Cochard et al. 1993) were purport-
edly achieved in Ivory Coast and Indonesia.
11.5.2 Cloning Improvement
The projected 30% yield increase by cloning the best individual palms from
commercial mixed hybrids provided the impetus in the development of the
tissue culture clonal propagation technique of the oil palm (Jones 1974; Hardon
et al. 1987; Meunier et al. 1988). Soh (1986), however, contended that the likely
increase would be about 13% with the first cloning, based on general theory
because of the low heritability for yield in advanced commercial hybrids. The
results from the CIRAD group comparing the improvements made by their
clones and improved hybrids from the MRRS programme were in general
agreement with Soh’s estimate (Nouy et al. 2006). However, the mean yield
advantage of clones over commercial hybrids summarized from five trials
testing 68 clones by AAR (Applied Agricultural Resources) was about 18%.
There were clones exceeding hybrids by 30% which could be recloned, but at the
same time hybrid improvement would have caught up by 10–15% (Soh et al.
2003a,b, 2006). Yields of up to 11–12 t/ha/yr oil have been reported in trial and
commercial clonal plantings (Mohd Isa et al. 2005; Soh et al. 2006).
11.5.3 Improvement in Other Traits
Besides breeding for yield per se, there are programmes which also emphasize
other desirable agronomic/economic traits, e.g. dwarfness or improved oil
quality. The semi-dwarf Dumpy.AVROS variety which was about 20%
shorter than the popular but tall AVROS variety was available since the
early 1980s and represented about 10–20% of Malaysia’s annual oil palm
plantings till recently (Soh et al. 2006; Mohd Isa et al. 2005). An improved
version of the Dumpy.AVROS variety, the Dumpy.Yangambi.AVROS
(Fig. 11.3) with better physiological traits and thus higher yield potential
has been developed (Soh et al. 2006). The development of dwarf palms high
in unsaturated fatty acids derived from their Nigerian E. guineensis accessions
by MPOB and E. oleifera E. guineensis hybrid-derived compact statured
clones by ASD have been announced (Rajanaidu et al. 2000; Escobar and
Alvarado 2003). Few groups have also stated to produce limited numbers of
340 A.C. Soh et al.
Fig. 11.3 Breeding semi-

dwarf high oil yielding
palms: Dy.Ybi.AVROS vs.
Dy.AVROS vs. AVROS
varieties
biclonal (clonal parents on both sides) and semi-clonal (clonal parent on one
side, usually the dura) hybrid seeds from proven parents. The impact from the
commercial planting of these newly developed special materials remains to
be seen.
Owing to the versatility of the crop in terms of the myriad uses of its oil, many
suggestions for various desirable traits to be improved in the palm and its oil
have been made. To reconcile opinions and lobbies, MPOB in 2003 organized
a workshop comprising breeders, agronomists, biotechnologists, oleochemists
and technologists, palm oil end-users and traders for prioritizing traits for
improvement in the oil palm, as it is inefficient if not impossible for breeders
to consider all the useful traits in a breeding program. The four top priority
traits in ranking order were: high oil yield, dwarf stature, resistance to Gano-
derma disease and high oleic acid oil (Table 11.2). The first three are agronomic
traits related to yield. This is not surprising as palm oil is still essentially
a commodity crop mainly used as food where high yield ensures lower cost of
production and competitiveness against other vegetable oils, e.g. soybean oil.
Except for high oleic acid content, other oil quality components and value
addition traits such as stearic acid, carotene or tocotrienols were given lower
11 Oil Palm 341
Table 11.2 Priority list (top four traits) of desirable traits for genetic improvement of oil palm
in Malaysia
Priority Trait Rationale
1 High palm oil Commodity crop, mainly used as food. Lower cost of
yield production. Oil already versatile in its uses.
2 Dwarf stature Current palm varieties grow too tall too fast. Inefficient
harvesting. Scarcity and high cost of workers.
3 Ganoderma Ganoderma basal stem rot is becoming a serious problem in
resistance young second cycle plantings, decimating the stand. Cultural
and chemical controls are ineffective.
4 High oleic acid Healthy monounsaturated oil. Liquid oil in temperate countries
for use in salad dressing and cooking. More suitable for
production of oleochemicals and biodiesel.
rankings. This priority list of traits is perhaps most relevant to Malaysia. In

African countries, drought resistance and Fusarium wilt resistance would have
higher priority, as with resistance to bud rot in some Latin American countries.
Difficulty of harvesting tall palms is currently not a constraint in Indonesia with
abundance of cheap labour.
11.6.1 Oil Yield
High palm oil yield is still the prime goal of most if not all breeding pro-
grammes. However, there are many facets in terms of achieving a high yield.
Potential yield. This is the maximum yield achieved by a variety when grown
under stress-free conditions to which it is adapted (Evans and Fischer 1999).
Modern high-yielding varieties, resulting from a plant ideotype breeding
approach are typically smaller statured plants that can be planted at higher
density, are consequently capable of high biomass production and possess a
high harvest index or better conversion of dry matter directed to yield rather
than to vegetative growth. The combination of high biomass production and
high harvest index results in a super high yield (Soh 2005). Such varieties are
single cross hybrids maximizing heterosis and stand uniformity. Early oil palm
breeding efforts were biased toward high early individual palm yield resulting in
aggressive plant types, and their use in mixed hybrid planting would not fully
exploit the potential yield of the crop. The ideotype approach in oil palm
breeding has been advocated since the early 1980s and some early results from
these efforts are available (Breure and Corley 1983; Squire 1984; Breure 1986;
Henson 1998; Soh et al. 2006).
Harvestable/recoverable/realizeable yield. Harvesting oil palm fruit bunches
particularly from older and taller (5–12 m) palms is still a tough and tedious
manual operation (man with sickle on long pole) with no imminent prospective
342 A.C. Soh et al.
mechanized alternative resulting in inefficient harvesting and crop loss due to

poor loose fruit recovery and low quality of unripe, over-ripe and rotten fruits.
Dwarf palms with thinner and longer bunch stalks would facilitate harvesting
manually or mechanically. Non-shedding fruits would reduce loss of loose
fruits (Osborne et al. 1992), while low lipase fruits would have longer ‘shelf-
life’ (inhibiting the production of free fatty acids) thus reducing the need for
more frequent harvesting rounds. Virescent fruits (green unripe, bright yellow
ripe) would facilitate identification of ripe bunches especially in tall palms
as compared to the common nigrescens fruits (dark purple unripe, orange
red ripe).
Adaptability Genotype by environment interaction (GE) is a function of
both differential genotypes and differential environments. If either compo-
nent is not differential, then a GE effect cannot be detected. This would
explain its non-detection in earlier oil palm studies (Rosenquist 1982; Cochard
et al. 1993). With current hybrid progenies and clones being more geneti-
cally uniform and discrete, and as such materials are being planted across
national borders and agri-ecological zones, GE interaction will assume
more importance, necessitating the breeding of genotypes with local adap-
tation (Donough et al. 1996).
Abiotic and biotic stress tolerance. Breeding for drought tolerance is integral
in the oil palm breeding programmes in West Africa, as there is a long drought
period annually (Houssou et al. 1987; Okwuagu and Ataga 1999). In Papua
New Guinea, oil palms planted on sandy soils tended to exhibit Mg defiency
and hence tolerance to Mg deficiency has been an important breeding objective
(Breure et al. 1986). Basal stem rot caused by the Ganoderma boninense basi-
diomycete fungus has become a serious disease in the major oil palm growing
countries particularly in young oil palm replantings from oil palm and coconut
where it has become more prevalent (Ariffin 2000; De Franqueville et al. 2001).
It is no longer a disease confined to high water table areas and affecting only
older palms (>15 years old). Cultural and chemical control measures are
cumbersome and ineffective. Disease tolerance is the long term solution requir-
ing an important separate breeding programme sometimes involving multi-
partite collaborative research efforts (CAB International 1988; Breton et al.
2005). Sources of tolerance genes have been found in MPOB’s Zaire Camer-
oon accessions (Idris et al. 2005) and in advanced breeding populations by
Durand-Gasselin et al. (2005). Nursery screening techniques have just been
developed requiring refinement (Ariffin et al. 1995; Breton et al. 2005). Oil
palm planted in West Africa not only needs to be drought tolerant but also
tolerant to vascular wilt disease caused by Fusarium oxysporum f.sp. elaedis.
Disease tolerance is available and its nursery screening is an integral part of the
breeding procedure (De Franqueville and Renard 1990). Lethal bud rot deci-
mates oil palm plantings in many parts of Latin America. OG hybrids appear
to be tolerant although the pathogenic cause of the disease is still contentious
(Ariffin 2000).
11 Oil Palm 343
11.6.2 Oil Quality
As palm oil’s principal use is in food production, its dietary quality is under
close scrutiny by the current health conscious consumers. Palm oil is often
regarded mistakenly as a saturated tropical fat with the connotation that its
consumption will lead to elevated levels of blood cholesterol and risk of cardi-
ovascular heart disease (CVD). Although it contains about 42% saturated fatty
acids, it is in the form of palmitic acid which is not known to be cholestrolemic;
unsaturated fatty acids such as the monounsaturated oleic acid (40%) and
polyunsaturates (linoleic and linolenic acids, ca. 15%) mainly comprise the
rest. Oleic acid has been identified as a top priority trait for improvement
because it confers a healthy liquid oil similar to olive oil that is marketed in
temperate countries for salad dressing and as cooking oil. It also serves as a very
useful feed stock for other oleochemical and biofuel industries. Palm oil also
contains other useful organic components such as carotene (vitamin A), toco-
pherols and tocotrienols (vitamin E) as well as other antioxidants which not
only confer health (anti-CVD, anti-cancer) properties to palm oil, but can also
be isolated and spun-off in the health-care and cosmetics industries (Yusoff
2000; Khosla and Sundram 1996; Sundram and Chandrasekharan 2000).

The oil palm is a cross-pollinated perennial tree crop. Not surprisingly, it has
adapted breeding methodologies developed in maize, e.g. recurrent selection,
and animal breeding, e.g. sire (pisifera) testing, index and BLUP selection, and a
close temporal and genetic correspondence of commercial hybrid production
with each cycle of breeding (Soh 1999).
The major oil palm breeding programmes adopt either the modified recur-
rent selection method (MRS; Soh 1999) or the modified reciprocal recurrent
method (MRRS; Meunier and Gascon 1972). The former is practiced by
programmes particularly in the Far East linked to or influenced by the pro-
grammes of the Unilever plantations group, while the latter is adopted by
countries in West Africa and Indonesia advised by CIRAD.
In the modified recurrent selection scheme (Fig. 11.4), selection of Deli D
parents for further breeding and for mother palms in commercial hybrid seed
production is based on family and individual palm performances, hence Rosenquist
(1990) chose to name the method as FIPS (family and individual selection).
Tenera parent selection for further P breeding is also based on FIPS.
Being female sterile, P is selected as the male parent for DP hybrid seed
production based initially on its T sib performance in the TT family followed
by a DP progeny test by crossing it with a sample of usually 3–5 of the selected
344 A.C. Soh et al.
Fig. 11.4 Modified recurrent selection scheme in oil palm
D female parents in a nested mating design (NCM 1), i.e. top-cross or sire
testing. The resultant commercial mixed hybrid is akin to a synthetic in the
plant breeding literature (Simmonds 1979; Allard 1960) except that its sub-
sequent progeny seeds should not be saved for commercial planting. The
variability within the commercial mixed hybrids varies with the genetic diver-
sity and inbreeding status of the parents used. The main advantages of this
scheme are that more recombinant crosses and genotypes can be turned over
within shorter time and smaller space without the need for extensive progeny
tests, and that the genetic variability of the commercial hybrids would ensure a
good adaptability and reduced risk of genetic vulnerability. This breeding
scheme exploits only general combining ability (GCA), but not specific com-
bining ability (SCA). Also, the assumption that the additive or GCA effects
expressed within the parental DD and TT crosses will be reflected in the
DT/P inter-population hybrid yield performance may be untenable, espe-
cially when parents become more restricted and inbred (Soh 1999; Soh and
Hor 2000).
In modified reciprocal recurrent selection (Fig. 11.5), parents selected for
further breeding and for commercial hybrid seed production have been progeny
tested. Instead of DP progeny testing, a DT progeny test of the selected D
and T parents from the DD and TT families is done. To save time, selfs and
sibs of the parents undergoing progeny testing are made and planted simulta-
neously as the progeny test crosses. The best hybrid crosses are identified from
the DT progeny test. The best hybrid is then readily reproduced as commer-
cial DP hybrid using the Ds from the selfs of the D parent and the Ps from the
selfs of the T parent. This scheme exploits both GCA and SCA, and the
11 Oil Palm 345
Fig. 11.5 Modified reciprocal recurrent selection in oil palm
commercial hybrid material is a reproduction of the progeny tested hybrid.

Depending on the inbreeding status of the parents the commercial hybrids are
generally more uniform or near true F1 hybrids. The scheme encompasses a
within-hybrid improvement phase to further refine a particular hybrid combi-
nation and a recombination phase involving outcrosses to maintain genetic
variability for long term genetic improvement. The main disadvantage is the
large programme size requiring 500 crosses and 180 selfs to be planted over
600 ha and evaluated over 15–25 years in order to select the top 15% crosses to
be reproduced as 3–4 million commercial hybrid seeds (Soh 1999); the world’s
requirement of oil palm seeds is about 250–300 million seeds per annum. The
other purported disadvantage is the severe inbreeding depression limiting self-
ing of the parents to only one generation and thus inhibiting expanded produc-
tion of a particular hybrid. Hybrid maize breeding in the early years also faced
this problem but has since circumvented it by selecting segregants more tolerant
of inbreeding.
The backcross breeding method is usually applied in introgression pro-
grammes in which desirable traits from a relatively unimproved genotype are
incorporated into an otherwise recurrent host genotype. The AVROS and the
Dumpy.AVROS were developed in this manner, in the former the cross of
SP540T with Bangun was backcrossed to SP540T and in the latter, the dwarf
trait in the Dumpy Deli was introgressed into the AVROS by backcrossing (Soh
et al. 2006). A similar approach is adopted in the introgression of desirable traits
(oil quality traits, dwarfness, disease resistance) from the relatively wild or
346 A.C. Soh et al.
unimproved recent E. guineensis and E. oleifera introductions to advanced

breeding populations (Soh et al. 1999; Sharma 2000). It should be borne in
mind that for a perennial tree crop backcrossing (in the strict sense) to the
original recurrent tree is seldom possible but rather to the recurrent parental
progeny or population.
In recombinant inbred breeding, which follows an earlier proposal based on the
dominance theory of heterosis rather than on overdominance and epistasis
(Pooni et al. 1989), programmes to develop recombinant inbred varieties have
been attempted. In this approach, there is no necessity to separate the breeding
parents into heterotic groups, and any superior recombinant genotype can be
developed by single seed descent into an inbred variety. In theory, at least, one
could achieve an inbred D variety superior to the T hybrid which could be
propagated indefinitely by selfing. Others would rather pursue D and T recom-
binant inbred line development to produce second or advanced cycle hybrids as in
maize breeding (Bernado 2002), perhaps based on faster commercial exploitation
of every hybrid generation and proprietary right considerations (Soh 1987, 1999).
With breeding for clonal propagation in tree crops where vegetative propaga-
tion is possible, maximum genetic segregation is generated by crossing unre-
lated trees with complementary desirable traits. This is then followed by identi-
fying the superior segregant progeny tree or individual genotype and
confirming and fixing it through cycles of field testing and selection (Simmonds
1979; Tan 1987; Brown et al. 1988; Kawano et al. 1998). With the ability to
clonally propagate oil palm via tissue culture, it is tempting to adopt this
approach. That would require repeated cycles of cloning (from clones), the
feasibility of which is still uncertain in the oil palm (Soh et al. 2003a). This
approach would better be used as an adjunct to the main recurrent breeding
programme, especially at the early DP testing stage of the recombinant phase
where the T progeny palms are still genetically segregating appreciably.
Index and BLUP selection. Tree crop breeding as cattle breeding has to
contend seriously with space and time constraints and hence with the critical
choice of the few highly selected parents to go into the next cycle of breeding and
also to hybrid production. This would apply more so in the choice of parents or
ortets for clonal propagation. Ascertaining the breeding values of the parents
and the combination of multiple traits to select are thus important selection
techniques (Falconer and Mackay 1996). The selection index method for multi-
ple trait selection and for incorporating plot and family information was found
to be useful for selecting ortets in oil palm and could be extended to breeding
parents which are usually selected based on GCA for individual traits (Soh and
Chow 1989, 1993; Baudouin et al. 1994; Soh et al. 1994). The selection index is
constrained by the inability to obtain accurate estimates of genetic variance and
heritability. The BLUP (best linear unbiased prediction) technique developed in
cattle breeding (Henderson 1984) can integrate unbalanced data from mating
and experimental designs and even production data and was used to obtain sire
(P parent) breeding values in oil palm (Soh 1994). BLUP has also been used to
11 Oil Palm 347
predict hybrid performance in oil palm (Purba et al. 2001), as in maize

(Bernardo 1994, 1995, 1996) and sugarcane (Chang and Milligan 1992a,b).
Breeding system. The oil palm is monecious bearing male and female inflor-
escences on the same palm occurring in different but overlapping cycles. Stress
conditions such as drought, malnutrition or plant competition favour male
inflorescence production (Corley and Tinker 2003). Pisifera palms do produce
female inflorescences especially under good growing conditions, but they
usually are aborted. Cross pollination in the oil palm is effected efficiently by
the pollinating weevil, Elaidobius kcameroonicus, in its natural home in Africa.
Prior to the weevil’s introduction to the Far East, pollination occurred by wind
and thrips (Thrips hawaiiensis) which was less efficient. Stringent controlled-
pollination procedures had to be adopted after discovering that serious illegi-
timate pollination had occurred in the commercial hybrid seeds resulting in high
frequency of D contaminants in young commercial T fields soon after the
introduction of the weevils to the plantations in the Far East. In male and
female inflorescences, anthesis and receptivity occur about 2 months after
emergence or appearance. The pollen viability and receptivity periods of the
male and female inflorescences are about 3–5 days. The set fruit ripens, chan-
ging colour from dark purple or black to reddish orange, in about 5 months.
Controlled pollination. The formalin surface-cleaned male inflorescence is
isolated with a permeable terelene/canvas/paper bag about 7–10 days prior to
anthesis. The anthesized male inflorescence is harvested and air-dried for about
3 h in an oven, chamber (38–398C) or air-conditioned chamber; the pollen is
shaken out, sieved and further dried in filter paper envelopes at 38–398C in an
oven or desiccator till 6% moisture content and stored in test or specimen tubes
in a freezer. Properly processed pollen can retain its viability for 6 months up to
a year; freeze-drying is recommended for longer storage. Isolation of the female
inflorescence is done in exactly the same manner. Controlled pollination is
carried out when the female inflorescence is observed to be receptive, by puffing
a 1:10–1:20 pollen: talcum powder mixture through a hole made in one or more
plastic windows sown onto the isolation bag (Fig. 11.6). The bag is removed
after a month and the fertilized bunch allowed to ripen in about 5 months. As a
stringent quality control measure against illegitimate pollination, the inflores-
cence is discarded if there is any hole or tear in the bag or the presence of weevil
spotted inside the bag.
Seed germination. The oil palm seed is recalcitrant and requires heat treatment
at 37–388C and 17–19% moisture content for D seed (20–21% for tenera seed)
for 40–60 days for germination at ambient conditions after rehydrating to
21–23% moisture for D (27–28% for T seeds). Germination of very thin-shelled
T and shell-less fertile P seeds is erratic (Arasu 1970) and in vitro germination
(embryo rescue) is advisable.
348 A.C. Soh et al.
Fig. 11.6 Controlled pollination in oil palm
11.7.3 Field Experimental Techniques

11.7.3.1 Mating Designs
Biparental (BIP), nested (NCM1 or North Carolina Model 1), factorial
(NCM2) and diallel are the usual mating designs adopted in the parental
DD and TT/P within population crosses as well as the DT/P interpopula-
tion progeny test crosses (Soh and Tan 1983; Soh 1999). BIP is more favoured in
the parental crosses as it would allow more cross combinations to be tested
when estimates of combining ability are not critical. NCM1 and NCM2 matings
are commonly used in the progeny test crosses, the former favoured if GCAs of
the parents are of primary interest and the latter favoured if SCAs and specific
combinations are sought. BIP would also allow the most cross combinations to
be tested if the objective is to seek the best combination irrespective of whether it
resulted from GCA or SCA effects. Owing to the difficulty to obtain a complete
set of the desired matings (NCM1, NCM2) with a large number of parents,
balanced incomplete and inadvertent unbalanced designs are made. General
least squares (GLM) and maximum likelihood (REML or restricted maximum
likelihood, BLUP) methods are used to analyze such experimental data.
11.7.3.2 Field Experimentation

Experimental layout. The randomized complete block design (RCBD) is most
commonly used in field trials. Owing to poor seed germination in some crosses,
incomplete blocks commonly result and missing plot or GLM analysis is
11 Oil Palm 349
needed. Augmented designs (Federer et al. 2001) would also be useful in such
situations. Incomplete block designs, e.g. the balanced lattice (Cochran and
Cox 1957) to circumvent soil heterogeneity effects within large blocks asso-
ciated with large sets of crosses have been attempted in oil palm but were found
to be cumbersome and did not improve trial efficiency much (Soh et al. 1990).
Plot sizes of 10–20 palms (without border trees) planted in 3–6 replicates are
commonly used with the large plots preferred if between-cross differences
especially in growth habits are suspected. When the trial is replicated over
locations, 2–3 replicates per location would suffice. Soh et al. (1989, 1990)
found that a trial size of 6 replicates of 16 palm plots or 5 replicates of 20
palm plots is suitable to detect treatment differences of 15% with a trial
coefficient of variation of 10% under Malaysian conditions. Single palm plots
have been found to be not efficient unless highly replicated to about 100 times,
and they are also not experimenter-friendly for field visual appraisal. Single
palm plots in completely randomized design (CRD) would be useful in field
screening for disease resistance and for estimating between palm within progeny
variability in genetic studies.
Data collection. The following data are collected on an individual palm basis:
For vegetative growth measurements (Corley et al. 1971; Corley and Breure
1981), height, girth, leaf production, leaf area and weight measurements are
made annually. For fresh fruit bunch yield (FFB), number and weight of ripe
bunches are recorded in each 10 day harvesting round. Yield data is collected
from start of harvesting at about 3 years after field planting till about 9 years.
For bunch analysis (Blaak et al. 1963), bunches are sampled over the yield
recording period to determine bunch and fruit quality and oil content. Fruit
sub-samplings are made to estimate the ratio of fresh fruit weight in the bunch
(F/B%), the ratio of mesocarp weight in the fruit (M/F%), the ratio of kernel
weight in the fruit (K/F%) and the ratio of oil weight in the mesocarp (O/M%).
The product F/B M/F O/M gives O/B% (oil to bunch) and F/B K/F
gives K/B (kernel to bunch). For the determination of a progeny mean for O/
B% about 160 bunches from about 40 palms are sampled over the yield
recording period. For individual palm means usually more than 5 analyses are
needed. The following trait data can be generated from the above
measurements:
Vegetative growth: girth, height and height increment, total leaf production,
leaf area index, leaf area ratio, vegetative dry matter production (VDM).
Yield: bunch dry matter yield, BDM = FFB dry matter/fresh matter.
Bunch analysis: oil yield, OY = FFB O/B; kernel yield, KY = FFB K/B;
kernel oil yield, KOY = FFB K/B 0.5. These are reflective of the
commercial oil extraction, kernel extraction and kernel oil extraction rates
achieved in the mills. Total dry matter production, TDM = BDM +
VDM; bunch index, BI = BDM/TDM; harvest index, HI = BI 0.4.
All these primary yield, yield component and morpho-physiological traits,
either formally based on measured data or visually/intuitively scored, are taken
350 A.C. Soh et al.
in consideration in most oil palm breeding programs, besides oil quality and
stress resistance in others.
11.8 Integration of New Biotechnologies in Breeding Programmes
11.8.1 Tissue Culture for Clonal Propagation of Oil Palm
The commercial oil palm planting material being a heterogeneous mixture of

non-uniform hybrid progenies is genetically variable. Individual palms within a
commercial planting can yield considerably more than the field average. Repro-
ducing these superior individual palms by conventional hybrid breeding would
take more than 20 years. Thus the original objective of clonal propagation in the
oil palm is to short-cut this process by capturing elite individual palms from the
genetically variable planting material as clones and mass-propagate them for
large scale commercial planting.
Despite the early success in developing the tissue culture clonal propagation
technique and the subsequent expanded efforts in clonal propagation for trial
and pilot field tests, large-scale commercial propagation and planting of proven
elite clones until very recently have yet to take-off. To do this successfully, a
number of critical issues including somaclonal variation, cloning efficiency,
ortet (parent palm of clone) selection efficiency, recloning and the suspension
culture system need to be resolved or circumvented (Soh et al. 2003a).
Somaclonal variation in the oil palm occurs as mantled parthenocarpic fruits
resulting in bunch abortion and sterility (Corley et al. 1986). This has been the
main stumbling block for commercial in vitro propagation of oil palm for the
past 20 years and continues to be so for expanded scale commercial production
(Fig. 11.7). Susceptibility varies between and within clones, and the risk tends to
increase with extended culture, recloning or liquid culture. The current expla-
nation of the causal mechanism is that of an epigenetic change involving
methylation of the homeotic or flowering MADS box genes (Van der Linden
et al. 2005; Auyong et al. 2005). Putative markers were found to be clone specific
and thus of no general applicability.
AAR (Applied Agricultural Resources Sdn Bhd) pioneered the development
of the commercial gel culture propagation method through research into pro-
tocol development and refinement to improve the cloning efficiency with mini-
mal risk of the mantled fruit clonal abnormality and by cloning a package of
ortets. This method, although adopted by other laboratories for commercial
propagation, is still an inefficient process being hampered by the low percentage
of palms amenable to mass propagation, low plantlet production capability and
high labour and space requirement. Commercial propagation is achieved by
putting a large number of ortets into culture each time to circumvent the cloning
inefficiency and to hedge on clonal abnormality risk. This approach requires a
11 Oil Palm 351
Fig. 11.7 Somaclonal variation in oil palm: mantled parthenocarpic fruits (left), bunch
abortion and sterility (right)
continuous supply of superior ortets and compromises on the maximum yield

potential of clones achievable.
Ortet or individual palm selection is inefficient for oil yield improvement
because of its low heritability. Good clonal testing is mandatory to identify
outstanding clones. To exploit these outstanding clones as proven clones
recloning is needed. AAR pioneered in the feasibility of recloning contrary to
other earlier experiences with higher clonal abnormality risk. Recloning is still
constrained by the inefficiency of the gel culture system. AAR subsequently
developed and demonstrated the feasibility of the liquid suspension culture
system for both clones and reclones again contrary to the high abnormality
risk experienced by others. The advantages of the liquid system are its very high
capacity for uniform plantlet production and its amenability to automation
including the use of bioreactors. With these advances the technology for com-
mercial production and planting of proven superior clones is in place.
Nevertheless, the ability to scale up clonal production with minimal soma-
clonal variation to capture a significant proportion of the world’s large hybrid
seed market (ca. 250 million) poses the biggest challenge (Soh et al. 2006).
11.8.2 Tissue Culture Process

The tissue culture cloning process in oil palm (Wong et al. 1997; Soh et al.
2003a, 2006) involves the following stages (Fig. 11.8) in a commercial labora-
tory (Fig. 11.9):
352 A.C. Soh et al.
Fig. 11.8 Oil palm tissue culture system (gel and liquid systems)
Fig. 11.9 Commercial oil palm tissue culture laboratory with two million plantlet production
capacity (Applied Agricultural Resources, S.B., Malaysia)
11 Oil Palm 353
Explant sampling. The explants are the young leaf pinnae of the youngest
non-emerged spear leaves of the selected palm. The harvested spear is surface-
sterilized before the leaves are unraveled in the laminar flow chamber and
1,000–2,000 explants of 1 cm2 pinnal segments are inoculated onto agar med-
ium in test tubes.
Callogenesis. Callus growth is induced on the explant using MS (Murashige
and Skoog 1962) medium which may be supplemented with additional nutri-
ents, variable levels and proportions of phytohormones, typically 2,4-D and/or
NAA and with or without the addition of charcoal. Some laboratories also
apply explant pretreatments. The explant cultures are incubated at ambient
conditions (26–288C, 90% relative humidity) in the dark or in lighted rooms.
Calli begin to appear on the leaf explants at about 3 months and continue
proliferation up to a year or more.
Embryogenesis. Callus differentiates into somatic embryos or embryoids
when nodular callus on explants is sub-cultured on medium with reduced levels
of hormones to stimulate callus differentiation into embryoids or somatic
embryos in air-conditioned culture rooms maintained at 26–288C and
50–60% relative humidity.
Embryoid proliferation and germination. Good embryoids are transferred
onto embryoid proliferation medium and sub-cultured usually at bimonthly
intervals. In the case of liquid suspension culture, embryogenic calli are trans-
ferred to liquid medium in a conical flask on an orbital shaker for proliferation.
At each monthly sub-culture, embryoids of the desired size are sieved out and
transferred to fresh medium.
Plantlet regeneration. In the gel system, shoots begin to germinate at about
the 5th sub-culture on the polyembryogenic mass consisting of secondary
embryoids, primary embryoids and callus. The shoots are harvested from the
polyembryogenic mass and put onto a liquid medium in a test tube for shoot
development and root induction. The polyembryogenic mass is put back onto
fresh medium for further proliferation. In the liquid suspension system, mature
embryoids are inoculated onto plated medium for germination. The shoots are
subsequently transferred individually into tubes for growth and root induction
as for the gel system.
Acclimatization. Rooted plantlets of about 8–12 cm height are selected and
planted out in trays containing inert potting mixture, e.g. sand, peat fibre,
vermiculite in a plastic incubation chamber in the conditioning nursery with
75% shade, 90% relative humidity and 27–308C temperature. Shade and
humidity are gradually reduced by opening the flaps of the chamber for increas-
ing lengths of time to condition the plantlets. After 3 weeks the trays of plantlets
are taken out from the plastic chamber and put under 50% shade to be
hardened and foliar fertilized. Hardened 3–4 leaved plantlets are harvested,
cleaned of its potting medium and treated with a fungicide before dispatching as
‘bare root seedlings’ contained in moistened plastic bags packed in carton boxes
to the plantation nurseries, where they are raised as for normal seedlings.
354 A.C. Soh et al.
11.8.3 Commercial Planting of Oil Palm Clones
The commercial planting of oil palm clones, totaling less than 20,000 ha out of
the world’s total oil palm planting area of 14 million ha, can be considered in its
infancy or at only a larger scale pilot commercial testing state (Fig. 11.10). The
clones are produced by a handful of commercial tissue culture laboratories with
Fig. 11.10 Nursery and field plantings of oil palm clones

11 Oil Palm 355
only a couple dominating the production, although more laboratories have

been set up lately. The clones are usually derived from ortets selected from D
P/T progeny test trials or mass selected from commercial fields with the former
preferred. Depending on the laboratory, reclones (reproduced from proven
clones) and clones from liquid suspension cultures may also be included. Pre-
ferably, a package of 5 different clones is to be planted at the same time to hedge
against abnormality (somaclonal variation) and inefficient yield selection risks.
To ensure adequate pollination for plantings on good areas, inclusion of low sex
ratio (lower female inflorescence production) clonal or hybrid palms in the
clonal package is usually practised.
11.8.4 Clonal Fidelity and Performance Tests

As evident from the above there are still a number of unresolved issues in both
the commercial production and planting of clones, especially related to cloning
efficiency, clonal fidelity (abnormality risk) and yield selection efficiency.
Hence research is still ongoing to develop and fine-tune protocols in cloning,
recloning, gel or liquid cultures, and techniques in selecting and testing ortets
and clones as well as planting system configurations to maximize the yield
potential of clones in the field (Soh et al. 2003a). For fidelity testing, from
each clone/protocol treatment about 40–100 plantlets may be sampled repre-
senting different levels of plantlet production and planted in the nursery and the
field at closer spacing to observe for clonal fidelity, i.e. for normal or abnormal
behaviour (e.g. mantled abortive bunches, sterile palms, abnormal vegetative
growth, terminal inflorescence or premature flowering in the nursery). Plantlets
from proliferating cultures from good ortets and treatments are also planted in
clone test trials and commercial test plantings (which may include different
planting arrangement treatments) for observing yield performance and field
management efficiency besides clonal fidelity.

Molecular markers are now widely used in plant breeding, and oil palm breed-
ing is no exception since the oil palm is a perennial tree crop where savings in
breeding time, space and effort are much sought after. DNA from oil palm is
obtained mainly from leaves, although it is also possible to isolate it from other
tissues (Lim and Rao 2005). The detection and determination of markers in
oil palm is possible through molecular biology techniques such as isozyme
(Rajanaidu et al. 1993; Choong et al. 1996), RFLP (Cheah 1990; Mayes et al.
1996), RAPD (Shah et al. 1994), AFLP (Cheah 2000; Kulratne et al. 2000) and
SSR-PCR (Billotte et al. 2001, 2005; Chua et al. 2005; Phoon et al. 2005).
RFLP, AFLP and SSR markers are particularly robust, reliable and were
356 A.C. Soh et al.
mainly used to saturate the majority of oil palm genetic maps (Mayes et al. 1997;
Billotte et al. 2005; Ting et al. 2005).
Some of these polymorphic markers are used for oil palm DNA fingerprint-
ing, particularly useful for genotype identification and genetic diversity assess-
ment (Cheah and Wooi 1995; Cheah et al. 1999). As concerns about breeder’s
rights on planting materials increase, Malaysia has gazetted the Plant Variety
Protection Act in 2004. The Malaysian Department of Agriculture aided by
MPOB and industry members is in the process of drafting test guidelines for the
conduct of tests for distinctness, uniformity and stability in compliance with
UPOV (Union for the Protection of New Varieties of Plants). DNA fingerprint-
ing for genotype identification is identified as one of the elements to comple-
ment morphological markers. Of particular interest are elite clones and inbred
parents for hybrid seed production that need protection from piracy and sub-
sequently ensure profit return to the rightful breeder. Discriminating commer-
cial ‘varieties’ of oil palm is envisaged to be more difficult as they are mixed
hybrids from parents with very similar genetic backgrounds.
In advanced breeding programmes, it is becoming increasingly difficult to
select for extreme outliers because of the highly selected nature of the genetic
materials. Genetic diversity studies with the estimation of genetic distance or
relatedness between oil palms can help identify suitable divergent parents for
base broadening breeding or for crossing to enhance hybrid vigour in the
progeny or seed produced. The same can be applied to inbreeding programmes
to assess homozygosity or inbreeding level of the resulting materials and in
backcrossing programmes to recover the recurrent genotype. Genetic diversity
studies can assist in hastening the prediction of parents with good combining
ability in that a pre-selection of materials can be included in a breeding trial to
expedite creation of homozygous inbred parents.
The ability of molecular markers to assess relatedness also introduces the
possibility of assessing illegitimacy of a cross or a commercial hybrid (Corley
2005). With increasing efforts and expenditure invested in oil palm breeding
programmes and commercial field planting of hybrids, illegitimacy in the
breeding and commercial hybrid materials cannot be tolerated. With molecular
marker technology, crossing programmes can be quality-checked and breeding
as well as commercial hybrid seed production will become more efficient.
With the construction of genetic maps, it is possible to identify the genomic
location of specific traits by using genetic markers. Marker based selection can
be used as an early screening technique for specific traits of interest such as
desirable fatty acids, amenability to tissue culture, fruit colour, shell thickness,
stem height and mature frond length, even before the palms mature or before
the trait is expressed. Marker assisted selection can help reduce the breeding
trial size thus increasing the chances of selecting desirable extreme outliers for a
given trait and thereby expedite the improvement process. Single genes control
fruit colour and shell thickness, therefore single markers can be developed for
their selection (Mayes et al. 1996; Moretzsohn et al. 2000; Billotte et al. 2005).
The issue at hand is whether the marker is close enough to the target gene to
11 Oil Palm 357
prevent recombination and thus is reliable. For a marker for virescent fruit it
appeared to be so, but not for the shell gene. Other traits mentioned above are
likely to be controlled by multiple genes or quantitative traits and several
markers are needed for their selection. Markers controlling quantitative traits
can be found in the same or across different linkage groups. The location of
these quantitative trait loci (QTL) can be identified using fine mapping techni-
ques, such as the bulked segregant analysis (BSA) as described in Weising et al.
(2005). Currently, the interest is to analyze single nucleotide polymorphisms
(SNPs) to further saturate the genetic map so that more precise markers may be
located (Rajinder and Cheah 2005).
Fluorescent in situ hybridisation (FISH) was used to detect collectively the
amount of E. oleifera’s DNA in interspecific crosses of E. oleifera (O) with E.
guineensis (G). The whole genome of O can be labelled, hybridised to the O G
genome and detected with fluorescent dye. Chromosomes that contain genome
from O will show fluoresce under fluorescent microscope revealing the compo-
sition of genetic material from both parents. The inheritance of genetic material
in the F1 hybrid is 50% from each parent. However, as reintroduction of
desirable G characteristics by backcrossing with G continues, percentages of
genetic content from O would gradually reduce from 50 to 0%. Monitoring
with FISH therefore enables targeting of progeny with the desirable genetic
composition and reduces the number of backcrosses needed (Maria et al.
1998a,b; Cheah et al. 1999) prior to screening for other desirable traits (e.g.
oil quality), which is essential in long life cycle crops.
As alluded to earlier, the current thought on the cause of the floral abnorm-
ality somaclonal variant is that of methylation (Jaligot et al. 2005) and alteration
in the expression of MADS box gene (Van der Linden et al. 2005). In order to
detect methylation in the MADS box gene, use of methylation sensitive MADS-
box directed profiling is currently under investigation (Auyong et al. 2005).
There has been also an increasing interest in the expression marker system
for the detection of complicated biological events such as embryogenesis and
floral abnormality. These markers should detect expression level changes coin-
ciding with a biological event, making it possible to detect an event of interest.
Genes involved in embryogenesis have been isolated from cDNA libraries
(Ong-Abdullah et al. 2005). Currently, more genes are to be analysed and
identified using the microarray technology (Low et al. 2005), which can screen
through thousands of expressed genes in one single analysis. Several genes
involved in floral abnormality and embryogenesis have been identified and
are undergoing expression studies and functional analysis.
Genetic transformation of oil palm via biolistics is now a reality with
BASTA-resistant oil palm produced (Ghulam Kadir et al. 2005) and used as
a selection means for transformation. Of particular interest is the creation of
oil palms that produce desirable fatty acids. This can be done by enhancing
and/or suppressing key genes that produce certain fatty acids. Some of these
key enzymes are: b-ketoacyl-ACP synthase II (KASII) which catalyses con-
version of palmitic acid to stearic acid, stearoyl-ACP desaturase which
358 A.C. Soh et al.
catalyses conversion of strearic acid to oleic acid, palmitoyl-ACP thioesterase

and oleoyl-ACP thioesterase which cleaves palmitic and oleic acid respectively
from acyl carrier protein (ACP). Other novel genes of interest to be inserted
into oil palm are bioplastics producing genes. These genes’ expression can be
targeted to leaves (e.g. bioplastic genes) or mesocarp (fatty acid synthesis
genes) using suitable promoters. Nursery plants of such putative transfor-
mants are available awaiting field testing for stable trait incorporation and
expression as well as in biosafety requirements. Research in Agrobacterium
transformation has also been initiated in view of the inefficient transforma-
tion process associated with the biolistics approach yielding chimaeras and
partial or multiple copies of the transgene.
11.9 Commercial Seed Processing
The various steps of oil palm seed processing are described according to
Periasamy et al. (2002):
Depericarping. The ripe bunch from controlled pollination harvested from
the D mother palm is chopped to separate the fruit spikelets which are then
allowed to ret for days in a basket or gunny/raffia sack to detach and soften the
fruit. The mesocarp of the fruits is then stripped off using a depericarping
machine to give the fresh seeds. Fresh seeds are then further cleaned of the
adhering fibres, washed with a detergent and treated with a fungicide.
Fresh seed quality checking. A sample of the seeds is taken from each bunch,
the shells cracked and the kernels and embryos scored for quality. The kernels
must be present, fresh looking and not diseased or rotting. The embryos must be
well-formed and not shrivelled, cylindrical in shape and white in colour with a
pale yellow green tinge. Bunches with more than 80% good quality seeds are
acceptable for further processing into commercial seeds.
Seed moisture adjustment and storage. A sample of seeds from each bunch is
checked for its moisture content. The seeds from the bunch are then soaked or
surface air-dried to achieve 18–19% moisture content for storage in an air-
conditioned room at 218C.
Heat treatment. Stored fresh seeds (at about 18–19% moisture) when
required to be germinated are sent to the germinator (hot room) to be heat
treated at 37–398C for about 50 days. The heat treated seed can be directly
processed for seed germination or cold stored again as ‘preheated’ seed.
Resoaking and germination. Preheated seeds need to be remoistened to
21–22% moisture before setting them to germination at ambient conditions.
Germination begins after a week with weekly flushes for about 6 weeks. With
good quality seeds, more than 90% germination rates would be achieved by the
third flush.
Seed selection and dispatch. Germinated seeds/seedlings with pearly white
plump and balanced plumules and radicles of <5 cm length are selected and
11 Oil Palm 359
collected in plastic bags with 200–250 seeds in each. These are then packed in
polystyrene-lined and bead buffered carton boxes for dispatch. With current
ease of air and surface transport systems, commercial oil palm seeds are now
exclusively dispatched as germinated seeds all over the world instead of pre-
heated seeds.
11.10 Oil Palm Seed Market
The world trade in oil palm seed is about 250–300 million seeds worth US$ 100
million at around 50 US cents per seed. The main supply and demand is in
Indonesia with about 117 million seeds, Malaysia with 85 million seeds and all
others with 85 million seeds as well. The break-down of the oil palm seed
suppliers, many of which are plantation-based companies and their estimated
capacities are given in Table 11.3.
The high crude palm oil prices boosted by oil demands for biofuel have
prompted the opening of more land in Indonesia and other countries for oil
palm cultivation resulting in increased demand for oil palm seeds, and conse-
quently many seed companies have stepped-up production. The supply of oil
palm clonal planting material from about 6–8 tissue culture laboratories is still
Table 11.3 Oil palm seed production companies and their estimated annual seed supply
Estimated supply
Country Company (in million seeds)
Indonesia Indonesian Oil Palm Research Institute (IOPRI) 35
PT. Socfm Indonesia 20
PT. London Sumatera Indo 20
PT. Bina Sawit Makmur 20
PT. Tunggal Yunus Estate 6
PT. Dami Mas Sejahtera 10
PT. Tania Selatan 6
Malaysia Felda Agricultural Services S.B. 27
Guthrie Research Chemara S.B. 20
Golden Hope Research S.B. 10
United Plantations Bhd. 8
Applied Agricultural Resources S.B. 6
Ebor Research, Sime Darby Plantations Bhd. 3
EPA 3
Sawit Kinabalu 3
Others 8
Costa Rica ASD 20
Papua New Guinea Dami 20
Africa Ivory Coast, Zaire, Nigeria 17
Thailand Univanvic 5
South America Colombia, Brazil, Ecuador 14
360 A.C. Soh et al.
very small constituting less than 1% (ca. 2 million) of plants and may rise to
5–10 million in 5 years time; the high price and limited supply of cloned plantlets
is attributed to the still inefficient commercial tissue culture process.
11.11 Concluding Remarks
At a still rapidly increasing population particularly in the developing countries,

food oil consumption is expected to rise both due to increased population and
rising affluence. Additionally, there is an increasing demand for vegetable oils
to produce biofuels in response to high fossil fuel prices and environmental
concerns on the continuing dependence on mineral oil. Palm oil being the
cheapest oil that can be produced sustainably is likely to meet the bulk of this
need. Oil palm plantations are likely to increase appreciably and existing old
plantings will be continuously replaced with new improved varieties. As such,
oil palm breeding and its research will continue to expand utilizing both con-
ventional and biotechnological approaches.
Acknowledgements We wish to express our sincere thanks to our company, Applied Agri-
cultural Resources Sdn. Bhd. and its principals, Boustead Plantations Bhd. and Kuala
Kepong Bhd. for permission to publish this paper.
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Chapter 12
Coconut
Lalith Perera, Suriya A.C.N. Perera, Champa K. Bandaranayake,

and Hugh C. Harries
12.1 Introduction
The coconut palm, Cocos nucifera L. is now grown mainly by smallholders but
was once the major perennial plantation oil crop widely cultivated in the humid
tropics. It has a pan-tropical distribution, occurring in coastal areas between the
latitudes 208 north and south of the equator and at altitudes between sea level
and 1,200 m. Coconut grows best under conditions of high humidity, at
temperatures of 27–308C and on moderately to well-aerated soils.
Coconut is the most extensively grown and used palm in the world and about
10 million families in over 80 countries rely on coconut as their main source of
food and income. Coconut is a smallholders’ crop and a major proportion of
the production is usually consumed locally. The actual percentage of domestic
consumption of coconut in Asian and Pacific Coconut Community (APCC)
countries was around 64% in 2001. Coconut is mainly an oil crop, particularly
rich (48%) in lauric acid (Jones 1991). Virgin coconut oil (Bawalan and
Chapman 2006), expelled under low heat from fresh coconut meat to preserve
its natural vitamins and enzymes (Marikkar et al. 2007) is now becoming
popular in the pharmaceutical industry and is gaining a significant international
market. Coconut as a bio-fuel is also a newly emerging product which would
reinstate coconut palm as a valuable oil crop in the not too distant future.
Already people on the island of Bougainville in Papua New Guinea are
powering up their vehicles and generators with environmentally friendly
coconut bio-fuel. In addition, the coconut industry offers a wide range of
coconut products such as desiccated coconut, coconut cream, coconut milk
powder, defatted coconut, coconut fibre and fibre products, shell charcoal and
activated carbon, coconut vinegar and coconut arrack (liquor) etc. for the local
and the international markets. Tender coconut as a natural beverage is
presently gaining popularity and currently there is also a growing international
L. Perera (*)
Genetics and Plant Breeding Division, Coconut Research Institute of Sri Lanka,
CRISL, Lunuwila, Sri Lanka
e-mail: kanthaperera@yahoo.com

370 L. Perera et al.
market for fresh coconut water. Fresh coconut water is already widely used as a
natural beverage in some coconut producing countries, India being the largest
nut water consuming country whilst in Brazil, in the state of Sao Paulo alone, a
daily consumption of 100,000 nuts has been estimated. Accordingly there is a
vast interest in growing dwarf coconut varieties for water utilization. Coconut
oil is also the principal raw material used in the manufacture of soap, glycerine
and margarine. The lauric acid from coconut oil is used to manufacture deter-
gents, cosmetics and pharmaceuticals. Locally the trunk is made into furniture,
handicrafts and building materials, the fibre from the nuts for mattresses,
doormats and ropes. Coconut as a whole plant, particularly the colour forms
of dwarf varieties, are a tropical ornamental, much in demand as a signature
tropical landscape element (Meerow and Ayala-Silva 2006). Because of the
multiplicity of uses of the coconut palm, it is termed ‘one of Nature’s greatest
gifts to man’ (Burkill 1966) and also as ‘The Tree of Life’.
The estimated total area under coconut in the world in 2001 was 11.8 million
ha. The contribution of the major coconut growing countries in terms of land
area cultivated with coconut are Indonesia (31%), the Philippines (26%), India
(16%), Sri Lanka (4%), Thailand (3%), Tanzania (3%), Malaysia (2%), Brazil
(2%), Papua New Guinea (2%), Vietnam (1%), Mexico (1%), and
Mozambique (1%). Samoa, Micronesia, Fiji, Solomon Islands, Vanuatu,
Palau, Bangladesh, China, Myanmar, Cocos Island, French Polynesia,
Guam, Kiribati, Tonga, Comoros, Ghana, Ivory Coast, Madagascar, Nigeria,
Tanzania, Colombia, Dominican Republic and Jamaica contribute the other
8% of cultivated coconut lands. More than one half of the coconut production
however comes from Southeast Asia, mainly from the Philippines and
Indonesia while almost one quarter comes from Asia, mainly from India
and Sri Lanka. The remainder comes almost equally from American, African
and Pacific regions (Asian and Pacific Coconut Community 1997).
Cocos nucifera L. is a member of the monocotyledon family Arecaceae

(Palmaceae) in the subfamily Cocoideae that includes 27 genera and 600 species
and is the only species of the genus Cocos. Coconut possesses a diploid genome
with 32 chromosomes (2n = 2x = 32).
There are conflicting theories regarding the origin and domestication of
coconut. A number of theories supported a New World origin of coconut
with subsequent dispersal to Asia and Polynesia (Guppy 1906; Cook 1910;
Ridley 1930). For example, the centre of origin of Cocoid palms, the closest
relative of coconut, is north–western South America. However, a theory for
Polynesian and Asian origin has been postulated (Child 1964, 1974; Burkill
1966; Purseglove 1985; Dennis and Gunn 1971) and both Fremond et al. (1966)
and Purseglove (1985) have provided convincing evidence of a Southeast Asian
12 Coconut 371
origin and Indo-Pacific domestication for coconut based on ethnological and

entomological evidence. Evidence from fossils and archaeological studies argue
for a South–West Pacific origin of coconut (Child 1974; Purseglove 1985) while
Indian fossils and the Madagascar forest coconut support an Indian Ocean
origin (Chiovenda 1921; Mahabale 1976; both cited in Harries 1995). However,
at present the origin of coconut is still not fully resolved but reports are
available that wild specimens of coconut have been found growing in natural
coastal forests in the Philippines and Australia supporting the theory that
coconut originated in the Western Pacific. A possible region for coconut
domestication was considered to be Melanesia, on the coasts and islands
between South East Asia and the Western Pacific approximately between
New Guinea and Fiji (Child 1964; Purseglove 1985) but more recently a
submerged continental region of South East Asia (Malesia) has been suggested
(Harries 1990, 2002). In an effort to explain the presence of close relatives of
coconut in the New World, Purseglove (1985) suggests that the ancestral palm,
which is likely to have had a fibrous mesocarp and be able to float and to
establish itself under suitable conditions, could have been carried by ocean
currents from South America to Polynesia in a similar route to that suggested
for sweet potato (Purseglove 1965, 1968), and coconut may have evolved from
that ancestor as a separate event in the Melanesian region. Harries (1978)
describes a possible evolutionary process for coconut based on natural selection
and evolution of large-fruited coconut from a small fruited progenitor. Gunn
(2004) provides the most recent review on the phylogeny of the Cocoeae
(Arecaceae).
Swaminathan and Nambiar (1961) suggested that dwarf coconuts (coconut
palm with short stature, see varietal groups) may have originated as a result of
inbreeding among tall coconuts as these show limited self-pollination, but this
was not supported by subsequent cytological studies by Raveendranath and
Ninan (1974). Purseglove (1985) states that dwarfs are probably mutations of
tall types. However, Harries (1978) suggests that some characters of dwarfs
(i.e. early germination, precocity, nut shape, different and bright fruit colours,
the proportion of husk and for some dwarf forms the high resistance to lethal
yellowing disease) indicate that domestication is more likely since dwarf popu-
lations could never survive in the wild (Harries 1995).
Coconut has been distributed to all parts of the tropical world including
Central and South America, East and West Africa, South and Southeast Asia
and the Pacific islands. From its putative centre of origin, coconut has been
disseminated both east and west by floating in the sea and by human dissemina-
tion (Ohler 1984), particularly by the ancient Polynesian sea voyagers, who
carried coconut as a source of food and drink on long sea voyages (Harries
1978). Whitehead (1976) also suggests that the spread of coconut from its
putative centre of origin to Central and South America was the end result of
the dissemination process and once established in a new vicinity, human activity
accounted for further dissemination beyond the initial distribution range.
It is accepted that the coconut palm has been present on the Atlantic coast of
Africa and South America and around the Caribbean for less than 500 years
(Child 1974; Purseglove 1972) as there were no coconuts in those regions at the
time of Columbus’s voyages. There is, however, evidence that coconuts were on
the Pacific Coast of Panama in pre-Columbian time (i.e. before 1492), either by
early Polynesians carrying them or by ocean currents naturally distributing
them. Harries (1978) observed a great similarity in West and East African
coconuts pointing to a common source of coconuts for those regions. Coconuts
in the East African coast were once thought to have either been brought there
by ancient Arab traders or to have floated from India in the tenth century where
they had grown for 2000 years, but according to more recent studies (Schuiling
and Harries 1992; Krain et al. 1993), the presence of coconut palms could
pre-date human activity. It is also possible that Malaysian sea-rovers, who
reached Madagascar in the first century AD, took coconut with them to
Madagascar and the Comoro islands (Lebrun et al. 1998). From there coconut
subsequently reached the coast of East Africa. Coconut reached Cape Verde,
West Africa only after 1500 AD through the Portuguese (Harries 1977; Purse-
glove 1985), probably from East Africa or India, and was then taken to the
Caribbean and Atlantic Coast of America. European explorers after 1492
contributed to further dissemination of coconut by transporting them from
Asia and East Africa to West Africa, the Atlantic coast of South America and
the Caribbean region (Purseglove 1972). Zizumbo-Villarreal (1996), reviewing
the history of coconut in Mexico, reported that the first introductions of coco-
nut to the Atlantic Coast of Mexico were from West Africa and the Caribbean
islands around 1549. He further reports that the introductions to the West
Coast of Mexico originated from Panama around 1539, from the Solomon
Islands around 1569 and from the Philippines from 1571 onwards by the
Spanish during the Spanish colonial period. Plantations were developed
throughout the tropics by the end of nineteenth century. Coconut populations
that are now considered endemic to the Atlantic Coast of Africa, America and
around the Caribbean region are basically the same as the coconuts in East
Africa, India and Sri Lanka (Harries 1977) while coconuts in the Pacific Coast
of America are related to the Pacific islands and Southeast Asian coconuts
based on fruit component analysis data (Harries 1978; Vargas and Blanco 2000;
Zizumbo-Villarreal et al. 2005).
The classification of coconut has been highly non-standardized, resulting in

different authors in different countries using different terminology of coconut.
However the major classification of coconut is based on stature and breeding
behaviour which groups coconut broadly into two groups or types: tall
(also termed typica) and dwarf (also termed nana), but Menon and Pandalai
12 Coconut 373
(1958) were quoting Narayana and John (1949) who included javanica as
another type of dwarf in India. Liyanage (1958), working in Sri Lanka, used
similar typica-nana terminology, but discarded javanica and described
aurantiaca as a new group. Neither of these South Asian classifications includes
the Niu leka dwarf from the South Pacific. Tall types are the most commonly
grown commercially exploited group and grow to a maximum height of about
20–30 m. They are predominantly allogamous (cross-pollinating), although a
limited degree of autogamy (self pollination) has been reported for some tall
groups (Bourdeix 1988; Bourdeix et al. 1990). The dwarf types in contrast attain
a maximum height of about 10–15 m and are predominantly autogamous.
Despite this higher degree of autogamy, dwarfs cross-pollinate with one
another and with talls. The major differences between tall and dwarf coconuts
are given in Table 12.1.
Table 12.1 Contrasting features of tall and dwarf coconuts

Character Tall (typica) Dwarf (nana)
Stature Tall (about 20–30 m) Short (about 10–15 m)
Bole formation at base of stem Yes No
Time till flowering 6–8 years 3–4 years
Economic life span Long (about 80–100 years) Short (about 40 years)
Bearing nature Continuous Seasonal
Nuts/palm/year Average 40 Average 80–100
Copra amount and quality 200 g/nut, good quality 80–100 g/nut, poor quality
Growing conditions Variable Sensitive to climate changes
Breeding habit Out-breeding In-breeding
In addition to the tall and dwarf groups, a few intermediate groups,

sometimes referred to as semi-talls or semi-dwarfs are also found. King coconut
in Sri Lanka (Liyanage 1958), and to the understanding of the authors,
Gangabondom in India (Menon and Pandalai 1958) and Niu Leka Dwarf in
Fiji (Powell 1868; Bourdeix et al. 2005a) are examples of such intermediate
groups. However the King coconut which has been classified as aurantiaca by
Liyanage (1958) is totally different from the Niu Leka, and they cannot be
classified in the same group.
As tall coconuts are predominantly cross-pollinated, they are highly
heterogeneous and consist of unique individual genotypes. However, many
coconut varieties (or cultivars) within the main group of talls exist that show
certain general morphological resemblance within the variety but very slight
dissimilarity or, in certain cases, contrasting dissimilarity between them. When
there is dissimilarity it is generally attributed to fruit traits (size, shape and
colour) and the proportions of the components of fruit (percentage weight of
husk, shell, nut water, meat etc.) though in certain instances traits such as soft
husk, sweet nut water, resistance to particular diseases (e.g. Vanuatu Tall
resistant to foliar decay virus and Sri Lanka Green Dwarf resistant to lethal
yellowing disease in Ghana) also contribute to the differences. These cultivars
often have different geographical origins and carry a prefix to the cultivar name
of the country of origin, or the region of origin within a country, where they are
originally and naturally grown. Earlier there had been no proper standardized
nomenclature for coconut, whereas now each variety is given a unique interna-
tional name, usually consisting of the group, whether it is a tall or dwarf, to
which a geographical or cultural reference is added; for varieties of uniform
colour, for instance, the different colour forms of the dwarf group, that colour
information is also added. Examples of cultivars are West African Tall, Sri
Lanka Tall, Mozambique Tall, Malayan Tall that originated from different
countries and San Ramon, Tagnanan Tall, Markham Valley Tall that origi-
nated from various regions within a particular country. Examples of such
cultivars in the dwarf group are Cameroon Red Dwarf, Sri Lanka Green
Dwarf, Malayan Yellow Dwarf and Madang Brown Dwarf (Madang is a
village in PNG). Furthermore, germplasm collections, field gene-banks and
the Coconut Genetic Resources Network (COGENT) database have certain
other entries termed as populations or accessions for the collections of coconut
populations within a particular coconut cultivar, identified in different geo-
graphic locations. Examples of such populations are Panama Tall Monagre and
Panama Tall Aguadulce, and West African Tall Mensah and West African Tall
Akabo, Sri Lanka Tall Ambakelle and Sri Lanka Tall Kasagala. They are also
sometimes referred to as ecotypes if the particular accession or population was a
result of an environmental selection over several generations. In these popula-
tions or accessions the morphological differences are either not noticeable or
very slightly detectable, but their adaptability for particular ecological niches or
for a particular pest or disease resistance cannot be predicted from morphology.
Within the main varieties, numerous groups of coconut do exist which
Liyanage (1958) referred to as forms of coconut and Bourdeix et al. (2005a)
referred to as variants that are phenotypically highly distinctive. Some examples
are Bodiri, Nawasi, Dikiri pol and Pora pol in Sri Lanka (Liyanage 1958),
Laccadive Micro Tall in India (Bourdeix et al. 2005b), Spicata in different
countries, Makapuno in the Philippines and Nim in Thailand.
Instead of the above varietal groups, Harries (1978) proposed two main
types of tall coconuts; one that has large, long, angular, thick-husked and
slow-germinating nuts with less free water content, the ‘Niu kafa type’ or wild
coconut, that evolved naturally and was disseminated by ocean currents, and
another that has more spherical nuts with an increased proportion of endo-
sperm, reduced husk thickness, early germination and resistance to disease, the
‘Niu vai type’ or domestic coconut, that was selected under cultivation for
increased nut water content and was disseminated by humans. Harries (1978)
further suggests that introgression of these two types and further selection and
dissemination by man gave the wide range of varieties and pan-tropical
distribution of coconut seen today.
12 Coconut 375
Whichever terminology is adopted, the authors are of the view that many of
the named varieties, cultivars or populations reported in coconut literature were
the result of either the vernaculars being used by local people and differences in
the regions where they come from or because of slight morphological differ-
ences. For example, Whitehead (1966) has shown that coconut palms in Pacific
islands are designated by several local names which do not always refer to
distinct cultivars, but to small morphological differences. It is also possible
that there could be some duplication of varieties in different coconut growing
areas that are known by different names and hence classified as different
varieties or populations. Therefore, until they are studied in detail by systema-
tic morphological and molecular investigations, the true differences between
varieties and populations remain unresolved.
In coconut, Cocos nucifera L., being the only species in the genus Cocos, there
are no closely related wild relatives known. Although it is now generally
assumed that truly wild type coconuts do not exist any longer, their characters
are present in modern populations. Buckley and Harries (1984), Gruezo and
Harries (1984) and Leach et al. (2003) reported wild types of coconut on
uninhabited coral atolls, on small isolated islands or on remote mainland
beaches. The domestic coconut is found associated with isolated or previously
isolated human settlements and dwarf coconuts can be included in the domestic
group, as they cannot survive in the wild. Present day coconut consists of a
mixed complex of wild and domestic characteristics, depending on which of
these forms predominated when cultivation began in each region.
Many different coconut genetic resources have been described by different
authors (Narayana and John 1949; Gangolly et al. 1957; Menon and Pandalai 1958
and references therein; Liyanage 1958, Bourdeix et al. 2005a). There is a great
diversity in fruit characters within and between populations for size, shape and
the colour of the fruit and proportions by weight of fruit components viz. husk,
shell, endosperm and water. Ashburner et al. (1997a) conducted a survey on
diversity in fruit components of South Pacific coconut, and reported great diversity
for fruit morphology in a range from populations exhibiting wild type characters to
populations displaying domestication characters. Variation in fruit shape varies
from near-spherical to short and long angular with different degrees of expression
of a pear shape (Foale 1991). It is also observed that there is a great deal of
variation in frond morphology, orientation of the crown, number and length of
bunch, number of female flowers and number of nuts per palm even within
populations (personal observations of the authors). It has been reported that
there is more morphological diversity in Southeast Asia than there is in South
Asia, Africa or South America (Benbadis 1992; Whitehead 1976). As a conse-
quence, more named varieties are found in Southeast Asia relative to other areas.
Coconut genetic resources are currently threatened by a high rate of

genetic erosion all over the world, mainly due to industrialization,
urbanization, infra-structure development, changing use of agricultural
land for high-value cash crops, and natural disasters such as cyclones,
tsunamis, droughts, pests and diseases. Moreover, coconut development
programmes involving the replanting of existing areas in regions of
greatest diversity with fewer numbers of high yielding hybrids or improved
varieties with a narrow genetic base threatens to displace older populations
resulting in a further reduction of the genetic base. Therefore, collection
and conservation of coconut biodiversity was nationally and internation-
ally recognized as an important objective, as future breeding is based on
collected material. As a result, the Coconut Genetic Resources Network
(COGENT) was established in 1992 as a global network under the auspices
of International Plant Genetic Resources Institute (IPGRI) with the
objective of strengthening national programmes to conserve and utilize
coconut genetic resources (http://www.cogentnetwork.org). Under this pro-
gramme, COGENT, with funding from the Asian Development Bank
(ADB) assisted by 13 Asia-Pacific countries in 1996 initiated the collection
of coconut genetic resources and in 1997 extended support to Mauritius,
Madagascar, Seychelles, Sri Lanka and Vietnam to collect and conserve
threatened or useful coconut biodiversity. The COGENT participants also
set up an international Coconut Genetic Resources Database (CGRD) in
1992 with the objective of construction of a computerized catalogue of
accessions representing a large number of coconut cultivars spread
throughout the coconut growing regions in order to gain an
understanding of coconut diversity and thereby promote coconut germ-
plasm exchange (Hamelin et al. 2005). The CGRD database was designed
to provide passport, characterization and evaluation data for the coconut
accessions in the database, and the number of accessions in the database
by the year 2003 increased to 1426 compared to 500 in 1994. These 1426
entries included 599 tall cultivars, 111 dwarf cultivars and 1 semi tall
cultivar, some cross-pollinating dwarfs and populations within cultivars.
However, results obtained by molecular studies on 33 coconut populations
in Sri Lanka, revealed a very low level of population differentiation (2%)
indicating very close relationships between those populations (Perera et al.
2001). This poses the question whether same genotypes carry different
accessions/codes in the collection, and this has become an issue to be
resolved urgently. The recent book on ‘Coconut Genetic Resources’
(Batugal et al. 2005) published by IPGRI and the CGRD database provide
a comprehensive coverage on the coconut genetic resources available
throughout the world. COGENT has also initiated the establishment of
four large multi-site international coconut gene-banks in Indonesia, India,
Papua New Guinea and Ivory Coast as well as 28 national genebanks in
24 countries, in addition to the existing small international collections in
Tanzania, Ivory Coast, the Philippines and India.
12 Coconut 377
As in many crops since early agricultural times, the coconuts grown all over the
world were derived by mass selection and open pollination, using criteria
determined informally by farmers themselves. The earliest selection of coconut
dates back 8,000–14,000 years (Harries 2002) during which time coconut had
been selected and domesticated for large round fruits rich in water as a source of
sweet uncontaminated water for seafarers travelling from island to island.
However, after commercialization of coconut during the nineteenth and
twentieth centuries, the yield of copra per palm or one of its correlates have
been the major criteria for selection of seed palms by farmers. The efficiency of
mass selection of mother palms based on desirable characters has been studied
extensively, and the progeny trials established in Sri Lanka occupied the most
prominent place in early coconut breeding research and generated much
information for developing criteria for selection of seed coconut palms. Most
prominent of these is the estimation of heritability values for a number of useful
characters in coconut (Liyanage and Sakai 1960) as effective criteria for
selection of seed palms (Liyanage et al. 1988). The progenies resulting from
open pollinated seeds are the basis of an improved population. For instance, the
response to selection for de-husked nut weight by open pollination was a yield
gain of 14.4% by selecting the best 5% of the population (Liyanage, 1972).
Current seed palm selection criteria in Sri Lanka and elsewhere are developed
from such observations. Early studies on coconut also provided information on
some useful correlates between seed characters, period to sprouting and
flowering, and initial yield and copra outturn. Seeds that sprout early promote
seedling height, leaf and root number leading to a shorter flowering period and
higher production of copra (Liyanage and Abeywardena 1957; Liyanage et al.
1988). Coconut nursery management practices all over the world adopt this
concept for culling weak seedlings from nursery beds, i.e. rejection of late
germinated seeds at a given period of time depending on the cultivar and
removal of weak seedlings again after keeping in the nursery for a fixed period
of time.
From the same progeny trials it was found that open pollinated progenies of
certain coconut palms are uniformly high yielding giving a mean yield of about
35–40% more copra than the population mean. That phenomenon was
explained as possessing of sufficient dominant yield traits in those palms to
pass on to their offspring despite having been indiscriminately pollinated by
unknown palms. Such palms were described as prepotent palms (Harland 1957).
However, their identification is laborious and time consuming and relatively
few palms prove to be prepotent from a large number tested, thus the quantity
of seed nuts collected from them for the industry is negligible. With the assump-
tion that progenies arising from artificial pollination using pollen of a prepotent
palm as the male parent will be equally high yielding as the natural progenies of
prepotent palms, seed production through artificial pollination of selected high
yielding mother palms from pollen of prepotent palms had been practised for
the genetic improvement of coconut. Raising an enormous quantity of
improved seeds demanded by farmers either by artificial pollination or stringent
mass selection is impossible, so seed gardens were designed specifically for mass
production of improved coconut genotypes. Since improved genotypes are
produced by controlled natural pollination the concept of isolated seed
gardens was well appreciated for mass production of superior genotypes
(Liyanage 1954, 1961a). The Sri Lanka Tall Sri Lanka Tall named as
Ambakelle Tall or CRIC60 from the Isolated Seed Garden (ISG) at Ambakelle
is an excellent example of such improvement attempts, which surpassed the
yield of ordinary Sri Lanka Tall coconut (Liyanage et al. 1988).
Despite inherent constraints, the inter-varietal hybridization has shown the
greatest gains in coconut breeding, demonstrating the usefulness of heterosis in
coconut. The beginning of scientific coconut breeding came when the first
controlled hybridisation was made in Fiji in 1926 between Malayan Red
Dwarf and Niu Leka Dwarf (Marechal 1928). In India, the first hybridisation
between tall and dwarf (West Coast Tall Chowghat Green Dwarf) was
attempted in 1930, with the intention of combining the quality of copra from
the tall parent and the high productivity as well as early flowering from the
dwarf parent. Sri Lanka initiated studies to test coconut hybrids in 1949 by
assessing the cross between Sri Lanka Tall and Sri Lanka Green Dwarf (Liya-
nage 1954, 1972; Liyanage et al. 1988) and the productivity of this hybrid was
remarkable – recording over 20,000 nuts per ha after 12 years from transplant-
ing (Manthriratne 1971, 1972, 1978). Most of the hybrid tests were conducted
between 1940–1960 and involved dwarf tall (inter-varietal) and tall tall
(intra-varietal) crosses and in these studies the superiority of dwarf tall over
local tall cultivars was well established. But it was not until the mid-1970s that
coconut F1 hybrids became widely available in commercial quantities. After the
first successful attempt (Harries and Romney 1974), many dwarf tall hybrids
have been produced utilizing different tall and dwarf cultivars originating from
different geographical regions. The crosses Malayan Dwarf Panama Tall
(Maypan), Malayan Yellow Dwarf West African Tall (PB121 or MAWA),
Cameroon Red Dwarf Rennell Island Tall (Maren), Malayan Red Dwarf
Tagnanan Tall (Matag or PCA15-2), Sri Lanka Green Dwarf Sri Lanka Tall
(CRIC65), Sri Lanka Green Dwarf San Ramon Tall (Kapruwana) are
examples of some of the most promising present day hybrids between dwarf
and tall used in a wide range of environments. More details on recommended
and preferred hybrids in different countries are described by Batugal (2005) and
Bourdeix et al. (2005b).
Recently it has been shown that tall tall hybrids, crosses between talls of
different origins are high yielding though they are not promising in terms of
early flowering (Bourdeix et al. 2005b). It must be noted that these crosses are
different to Sri Lanka Tall Sri Lanka Tall crosses which are combinations
between superior palms of the same variety. The Sri Lankan experience between
tall tall hybrids using Sri Lanka Tall San Ramon Tall was that they were
12 Coconut 379
highly promising in terms of copra per nut as well as total copra production per
unit area (Everard 2002) though they produced equally in terms of nut number
as local tall selection CRIC60, but less than 40% of nut number as the dwarf
tall hybrid CRIC65. Some of the other famous tall tall hybrids are West
African Tall Rennell Island Tall (PB213 or Waren) and West Afrian Tall
Vanuatu Tall (PB 214 or Wavan).
Although the first coconut hybrid tested in the world was a dwarf dwarf
hybrid (Marechal 1928), they are still the least exploited hybrids. However, the
authors strongly believe that dwarf dwarf hybrids are ideal trees for home
gardens in urban areas to grow them as ornamental palms while meeting the
daily coconut requirement if they show hybrid vigour for meat content per nut
and the quality of copra. In Sri Lanka currently there is a demand for coconut
varieties with short stature from people living in urban areas as palm height is a
problem in small home gardens and picking is difficult in tall varieties. Though
no follow up of Marechal’s work has been documented, a success story of a
cross between Malayan Yellow Dwarf and Malayan Red Dwarf varieties
(PB332) produced in 1971 at the ‘Marc Delorme’ centre in Ivory Coast is
reported by Bourdeix et al. (2005b), but no dwarf dwarf hybrids have yet
been widely distributed to farmers.
In terms of breeding for resistance to biotic and abiotic stresses, the inter-
crossing of resistant/tolerant germplasm with adapted high yielding materials
has been the strategy. All colour forms of Malayan Dwarf have been identified
as lethal yellowing disease resistant cultivars and the hybrid Malayan Dwarf
Panama Tall (Maypan) is therefore in particular demanded for areas where the
lethal yellowing disease phytoplasma occurs. Further, two Pacific coast tall
cultivars have been identified as highly resistant to this disease known as
amarilliamento letal in Mexico (Zizumbo-Villarreal et al. 1999). Other disease
resistant types include Vanuatu Tall, identified for tolerance to the coconut
foliar decay virus, and Sri Lanka Green Dwarf, for Cape St. Paul wilt tolerance
(also caused by phytoplasma) in Ghana. In India breeding for root wilt disease
is of high priority in the coconut breeding programme. Chowghat Green Dwarf
and Malayan Green Dwarf have been identified by the Central Plantation Crop
Research Institute (CPCRI), India as resistant varieties, showing a higher level
of resistance to root wilt disease compared to other coconut varieties. The cross
between Sri Lanka Yellow Dwarf Sri Lanka Tall has been identified as a
tolerant cultivar for Aceria mite by evaluating five commercially cultivated
coconut cultivars in Sri Lanka in a severely mite affected area (Perera 2005,
2006). Sri Lanka Yellow Dwarf and Gon thembili cultivars have also recently
been identified as tolerant cultivars to coconut Aceria mite (Perera 2006).
Drought is a serious constraint to coconut production in many countries as
coconut is mainly a rain fed plantation crop. Hence breeding for drought
tolerance has been given high priority in many coconut breeding programmes.
In Sri Lanka, a selection based on mean yield and genotypic adaptation to
changes in climate of the Sri Lanka Tall cultivar, correlating 15 years of
individual palm yield data with 15 years rainfall data has identified a new
cultivar released under the name of Ambakelle Special (Wickramaratne

1987a,b). In Ivory Coast PB-121 was identified as a drought-tolerant hybrid
(Bourdeix et al. 2005b), while much work has been done in India to assess and
measure the degree of drought tolerance, resulting in the identification of
several tolerant varieties (Rajagopal et al. 2005).
Studies on clonal propagation of coconut have been in progress in many
coconut growing countries and also in European laboratories since the 1970s,
but have not generated a protocol which can be applied to coconut breeding yet
(Oropeza et al. 2005). Successful in vitro culture of coconut embryos has been
developed as a tool for safe exchange of germplasm and to rescue embryos of
Makapuno coconut, a coconut variety which has high commercial value in the
confectionary industry but does not germinate when intact in the nut
(Carandang 2002; Rillo 2004).
Coconut breeding objectives are still primarily focused on high yield. The
definition of yield in terms of yield improvement in coconut is complex. It is
yield in terms of nut number that is attractive to the grower who sells coconut
on numbers basis whereas it is the size of the nut or the weight of meat or copra
that is demanded by the manufacturer or the processing sector. However, as the
number of nuts per bunch and size of the nuts is negatively correlated in
coconut, one cannot enhance both traits simultaneously by improving naturally
occurring coconut varieties by selection alone. On the other hand, in terms of
national production targets of a country it is the total meat or copra content per
unit planted area that is important. The breeding programmes aiming at a
higher nut number have succeeded through the production of dwarf tall
hybrids that surpass the yield of tall coconut cultivars by over 45%
(Perera 2005) when hybrids are agronomically well followed up. The low
copra content in the hybrid is more than compensated for by the number of
nuts produced. As copra content per hybrid nut is generally low compared to
tall nuts, at least in the context of Sri Lanka and India according to the
experience of the authors, hybrid nuts are less in demand by manufacturers
due to greater labour requirement during processing. In the contrary, the tall
tall coconut hybrid (CRISL98) in Sri Lanka is less demanded by the growers
though it produces about 40% more copra per nut, but is slightly lower in
number of nuts per palm. This is despite its producing more copra per unit area
than the dwarf tall hybrid compensating through the high copra content per
nut. It is a fancy hybrid for manufactures as well as for large scale coconut
growers who sell their nuts on a weight basis. Hence, the current breeding goal is
the development of hybrids that produce a large number of nuts carrying thick
kernel by carefully manipulating the parent palms in the breeding programme.
This has been achieved by the hybrid Kapruwana in which parents are large
12 Coconut 381
fruited San Ramon that originated in the Philippines and Sri Lanka Dwarf Green
which is a prolific bearer. Kapruwana produces as equally well as CRIC65 in nut
number and with high copra content per nut comparable with the hybrid CRISL98.
As the vegetative phase of the commercially grown tall coconut varieties is
long taking about 8–10 years, precocity in flowering is still a current goal in
coconut breeding. Significant progress has been achieved in precocity in the
coconut breeding programme by combining early flowering behaviour of
dwarfs with commercially grown tall coconut cultivars, but breeders still see
scope of shortening the time till flowering in hybrids by incorporating some
dwarf germplasm such as Salak Dwarf from Indonesia, which is exceptionally
early in flowering. Breeding for oil content or for an oil rich in a particular fatty
acid is in the early phase as an objective in coconut breeding.
With the global climate change, droughts have become frequent in many coco-
nut growing countries and hence they become a constraint in coconut production.
Therefore, in the recent past, breeding for drought tolerance has become a breeding
goal in the coconut breeding programmes of many coconut growing countries.
Selection of cultivars and individual genotypes in the fields prone to droughts has
been given high priority with the objective of selecting parents for breeding
programmes or improvement of local varieties through selection.
Though many major and minor coconut pests that damage coconut palms
exist, efficient and effective chemical and biological control methods are in
place to control them. However, all chemical and biological control measures
field tested so far under experimental conditions have either been unsuccessful
or not practical to adopt to control the coconut mite; Aceria guerreronis, a
microscopic coconut pest that lives beneath the perianth of the nut causing
damage to developing nuts. Hence breeding for tolerance to Aceria mite is a
current breeding goal in the coconut breeding programme in Sri Lanka and
India (Perera 2005, 2006). Screening of coconut varieties for tolerance to Aceria
mite is in progress and some initiatives have been taken to develop hybrids
involving crosses between tolerant parents (Perera 2006). Similarly breeding for
disease resistance has also been a current breeding objective given the situation
that in certain countries lethal disease of coconut destroys millions of coconut
palms. Among them, breeding for lethal yellowing disease in the Caribbean and
Latin American countries and for root wilt disease in India has been given
priority, where these phytoplasma diseases occur and spread.
Nowadays coconut farmers prefer to cultivate short stature palms because of
the unavailability of trained pickers or climbers and hence breeding for short
stature is also a current breeding objective.

The coconut is the sole species of genus Cocos and as such present and past
breeding work of this crop is limited to the intra-specific level. Furthermore the
long generation interval, high heterozygosity, the lack of a reliable method of
vegetative propagation and the limited number of seeds produced per year, all
limit the use of many traditional breeding methods employed in other crops.
Obtaining a pure line from heterozygous coconut remains an unrealistic
expectation because of the long vegetative phase. Thus coconut breeding is
confined to mass selection of phenotypically superior parent palms, and to
inter-varietal hybridization.
Mass selection is the most fundamental method for coconut breeding
(Liyanage 1955). Palms displaying superior agronomic traits such as stout
straight trunk with even growth with closely spaced leaf scars, well spread
crown with 25–30 healthy fronds, well packed with all stages of inflorescences
and developing bunches and palms with short bunch stalks are initially selected
from high yielding populations. Then the number of nuts produced per year,
average weight of husked nut and tolerance to adverse weather conditions and
to pests and diseases of the selected palms are assessed over a period of 3–5
years. Based on these data only the best 5–10% of the palms are selected as seed
palms and seeds collected from these palms are distributed as mother palm
seeds (Liyanage 1966).
However, as the number of seed nuts collected from mother palms was
insufficient to meet the growers demand, a programme called plus palm
selection was introduced in Sri Lanka in 1980 which was very much a
stop-gap measure to produce a seed that was, in quality, similar to a mother
palm seed, but was not exactly so. In adopting this programme, the actual
genetic quality of the seed may have been compromised to a degree due to the
extent of the seed requirement at that time.
Plus palms are superior palms selected from high yielding blocks of selected
estates based on good agronomic features, but quantitative data are assessed
just for a single harvest. The plus palms are selected in two stages: (a) Selection
of high yielding blocks from suitable estates based on yield figures for the past
five consecutive years, and (b) selection of plus palms within the high yielding
blocks. The high yielding blocks must satisfy the criteria that the minimum size
of the block should be 2 ha, mean block yield should be at least 8,400 nuts/ha/
year and the mean yield per palm should be at least 60 nuts/year, the block must
have at least 132 bearing palms/ha, the palms must be in the age range of 15–45
years, and the block must be free of pest and diseases. During the selection of
plus palms, 100 palms distributed randomly over the block are harvested, and
the mean number of nuts/palm of the block is estimated. Then the number of
nuts harvested from each palm is recorded and only the palms that record yield
above the estimated mean for the block are selected. Palms selected on the
above criteria and with satisfactory agronomic characters are then tested for
nut weight. Three ripe nuts are taken at random from each palm, husked and
weighed, and if the total husked nut weight of 3 nuts is more than 2.1 kg, they
are selected as plus palms. The harvesting of nuts of the marked plus palms is
done separately to avoid mixing of nuts in handling. The percentage of selection
following this method averages to about 20–24%. In order to obtain steady
improvement in the quantitative traits it is important to repeat the method
12 Coconut 383
adopted in mother palm selection and plus palm selection to their progenies
from generation to generation.
Since the gain from mass selection through open pollination is limited, seed
production by artificial cross pollination between high yielding parent palms
has been another breeding method in coconut (Liyanage 1954, 1966). Parents
are either selected based on phenotype or based on the progeny performance in
order to increase the response to selection. However, seed production through
this method is limited and therefore setting up of isolated seed gardens was the
answer (Liyanage 1961b). In these seed gardens natural assisted pollination
occurs between selected elite palms in current mass production of improved
coconut seeds. The isolation of the seed garden is achieved either by a forest
barrier around the seed garden that is thick enough to prevent pollen arriving
from outside (Liyanage 1955) or through 12 or more guard rows of the same
coconut variety. The latter is another technique widely practiced currently in
the establishment of seed gardens (Liyanage and Azis 1983). This technique was
designed after studying the movements of the honey bee, which is also a carrier
for the pollination mites.
For inter-varietal hybridization, different crosses between genetically diverse
varieties are made using hand pollination. Then the crosses are evaluated in the
field in comparison with recommended cultivars as controls, preferably in
different agro-ecological zones and in different soil types in order to select
new coconut hybrids and hybrids suitable for particular environments or soil
types. Once a suitable cross is identified, parent palms are multiplied in a seed
garden and seeds are produced by natural controlled pollination. The coconut
palm is monoecious and its inflorescence (a compound flower termed a spadix)
contains both male and female flowers. The inflorescence protected by a thick
sheath is called a spathe. The spathe naturally splits open to expose the
emerging inflorescence which consists of a central axis or rachis with up to 40
lateral branches. Each lateral branch is densely set with numerous (200–300)
male flowers and fewer (<50) female flowers. The principle of hand pollination
focuses on emasculation of the inflorescence followed by isolation of the female
flowers by means of a cotton bag (Liyanage 1954). When the female flowers
become receptive, selected pollen is artificially introduced. The close
supervision of emasculation determines the rate of success and the legitimacy
of the hybrid seed nuts.
Since production of seeds by inter-varietal crosses is laborious and time
consuming and generates only a very few seeds in relation to the general
demand for improved seeds, the isolated seed garden concept is also adopted
for production of inter-varietal hybrids. The two parent varieties, usually one
tall and one dwarf type or sometimes two tall types are planted in a given
proportion based on which variety is emasculated to serve as the maternal
parent. The mother parent is then emasculated and the female flowers of this
variety are allowed to be pollinated naturally by the other variety. Sometimes
only a single variety is planted in the seed garden where pollen collected from
another outside parent is blown on to the receptive emasculated inflorescences
of the palms in the seed garden. This method allows to change the hybrids
produced within the same seed garden by changing the pollen source.

Although conventional coconut breeding programmes using standard breeding
techniques based on phenotypic selection have been relatively successful, the
inherent constraints in coconut breeding as described previously make the
potential use of new biotechnologies highly attractive. Application of molecular
genetics in coconut breeding, particularly molecular markers began in the early
1990s and their application in coconut have been diverse, ranging from
assessing genetic diversity to creating genetic linkage maps. Initially the studies
were aimed at assessment of coconut genetic diversity and genetic relatedness at
the DNA level using universal marker techniques such as RAPD (Ashburner
et al. 1997b; Duran et al. 1997; Everard 1996; Dasanayake 2003; Dasanayake
et al. 2003), RFLP (Lebrun et al. 1998, 1999), AFLP (Perera et al. 1998;
Teulat et al. 2000) and ISTR (Duran et al. 1997; Rohde et al. 2000). Later on
the need for coconut specific markers was felt and accordingly in 1999 two sets
of microsatellite markers were isolated by two groups of scientists indepen-
dently using the cultivars Sri Lanka Tall (Perera 1999; Perera et al. 1999) and
Tagnanan Tall (Rivera et al. 1999). Microsatellites as co-dominant markers
have been particularly useful in analyzing highly heterozygous coconut for
genetic diversity and genetic relatedness estimates, germplasm characterization
and development of collections (Perera et al. 2000, 2001, 2003; Teulat et al.
2000; Dasanayake et al. 2003; Meerow et al. 2003), hybridity testing (Perera
et al. 2004), detecting somaclonal variation in coconut tissue cultures, and for
construction of genetic linkage maps (Herran et al. 2000; Lebrun et al. 2001;
Baudouin et al. 2006). A microsatellite kit comprising 14 primers and an
associated software for data analysis has also been developed (Baudouin and
Lebrun 2002) standardizing the techniques across laboratories for comparable
results, efficient detection of diversity and identification of varieties. More
recently DArT markers for coconut have been developed and used for diversity
studies (Perera 2005). Among these markers, SSRs have been the most widely
and extensively used in analyzing coconut.
12.8.1 Genetic Diversity Analysis
A high level of genetic diversity in coconut has been observed by Perera et al.
(2000, 2003) using microsatellites in a collection of 130 coconut individuals
representing 51 tall coconut varieties and 49 dwarf coconut varieties sampled
across the entire geographic range in the world. The mean genetic diversity
values based on Nei’s (1987) unbiased statistic observed by Perera (1999) was
12 Coconut 385
0.647 (0.139). Perera (1999) also found a reduced number of alleles in dwarf
coconut group compared to tall coconut group, comparable with a reduction in
the amount of genetic diversity in dwarfs. The results of Rivera et al. (1999),
who analyzed 20 coconut varieties, mainly from Southeast Asia and the Pacific
and Teulat et al. (2000), who studied 31 individuals of coconut comprising 14
coconut varieties were comparable with those of Perera (1999). Perera (2005)
also reported that heterozygous loci were evident not only in cross pollinating
talls (30%), but at lower frequency (2.5%) in dwarfs as well. The distribution of
genetic diversity between varieties within the tall group was observed to be
higher than that within the dwarf group. This finding has since changed the
germplasm collection strategies for dwarf and tall groups. The genetic diversity
study conducted for coconuts in Sri Lanka (Perera 1999) led to the finding that
the genetic base of Sri Lanka coconut is narrow. This has resulted in the change
of the breeding strategies of the Sri Lanka coconut breeding programme and led
to the importation of exotic coconut varieties and their incorporation in the
country’s breeding programme. Moreover, these studies identified redundan-
cies in the germplasm collection. Ashburner et al. (1997b) reported the use of
RAPDs to study the genetic diversity of 17 distinct South Pacific coconut
populations. They observed approximately 60% within population diversity
in general and noted two geographically cohesive groups and two single popu-
lations in a dendrogram. From the results, they concluded that there had been a
low but variable rate of gene migration between South Pacific populations with
possible founder effects and subsequent human selection. They proposed that
germplasm collection in the South Pacific region should focus on populations
rather than individuals because of the highly significant level of variation
observed between populations. Perera et al. (2001), who analyzed 33 coconut
populations belonging to Sri Lanka Tall variety using SSRs across the island
representing different geographical regions concluded that there was no popu-
lation differentiation (between population variation was only 5%) within Sri
Lanka Tall.
12.8.2 Genetic Relatedness
Perera et al. (2003) have constructed a phenetic tree diagram showing the
genetic relationships among 51 tall coconut varieties and 49 dwarf coconut
varieties across the world. Instantly this phenetic tree divided all tall coconuts
into two main groups, the first group comprising all the tall varieties from
Southeast Asia, the Pacific and the west coast of Panama and all dwarfs in a
sub-cluster within the tall cluster. The second group consisted of talls from
South Asia, East Africa and West Africa. Interestingly, none of the dwarf
coconuts grouped with the second main tall group. The results of Teulat et al.
(2000), based on cluster analysis according to UPGMA using the similarity
matrix based on the proportion of shared alleles, confirmed those of Perera
(1999). Results on genetic relationships based on microsatellite markers

generally agree with the results using other molecular techniques such as
ISTR markers (Rohde et al. 1995) or RFLP markers (Lebrun et al. 1998).
Rohde et al. (1995) studied 17 different coconut varieties from different geo-
graphical regions and identified groupings of African coconut with Indian
Ocean coconuts and Panama Tall coconut from the west coast of Panama
with Pacific and Southeast Asian coconuts. Lebrun et al. (1998) reported the
use of RFLPs in coconut from various geographical regions. Lebrun and
co-workers studied nine cDNA clones and one mitochondrial DNA (mtDNA)
clone (CoxI) from rice and one ribosomal DNA (rDNA) clone from wheat that
were hybridized to genomic DNA from 100 individuals of coconut palms repre-
senting 10 tall and 7 dwarf coconut varieties. Two main groups of coconuts were
identified, one comprising varieties from the Far East and the Pacific and the
other comprising varieties from India, Sri Lanka and West Africa. Higher levels
of diversity in Far East and Pacific coconut varieties were observed compared
with the other material. Clustering of Panama Tall originating from the Pacific
Coast of Panama with coconuts originating from the Pacific and the clustering of
West African tall with coconuts originating from the South Asia and Indian
Ocean group were also observed. These results were in good agreement with the
theory of Harries (1978) on the evolution and dissemination of coconuts. Results
from all these studies finally led to the conclusion that Cocos nucifera is divided
into two large genetic groups, the Southeast Asia and Pacific group and the
Indo-Atlantic group.
According to Harries (1978) naturally evolved coconuts, characterized as
Niu Kafa type, predominate in South Asia, West and East Africa, the
Caribbean and the Atlantic coast of Central America while coconuts selected
under cultivation, characterized as Niu Vai type, predominate in Southeast
Asia, some Pacific islands and the west coast of Central America. It is generally
accepted that the coconut palm has existed on the Atlantic coast of Africa,
South America and around the Caribbean region for only about 500 years
(Child 1974; Purseglove 1972), and that there is a great similarity between
these coconuts and those coconuts in East Africa, India and Sri Lanka
(Harries 1978). The grouping of Mozambique Tall, which Harries (1977)
suggested as the main original source of coconuts to West Africa and to the
Atlantic coast of America, with Cameroon Kribi Tall, West African Tall, Sri
Lankan Tall and Andaman Ordinary Tall from the Indian Ocean, appears in a
single cluster in the phenetic tree of Perera et al. (1999, 2003) and others
(Teulat et al. 2000) thus supporting the validity of Harries’s (1977) theory of
Portuguese-assisted coconut germplasm dissemination from the Indian Ocean
to the Atlantic after 1499. Interestingly, the variety Comoro Tall, from East
Africa falls in with the Southeast Asia/Pacific main tall group and it seems that
this variety originated from Southeast Asia coconuts, the Niu Vai type. Lebrun
et al. (1998) noted that the variety Comoro Tall took an intermediate position
between Southeast Asian coconut populations and the Indian Ocean
populations, based on RFLP markers. The Thailand Tall varieties, Thai Tall,
12 Coconut 387
Pak Chok, Talai Roi mainly group with the South Asia/Africa Tall group
while Kalok variety groups with the Southeast Asia/Pacific tall group. The
results of a detailed study on varieties of coconuts in Thailand by Harries et al.
(1982) based on fruit component analysis of coconut suggested that both Niu
Kafa type and Niu Vai type of coconuts are present in Thailand, with the Niu
Kafa type present on the Indian Ocean coast of the country. Harries included
the variety Kalok with other large fruited forms such as varieties San Ramon
and Tagnanan Tall in the Philippines, Bali Tall in Indonesia, Rennell Tall in
the Solomon Islands and Panama Tall and Peru Tall from the Pacific coast of
America. He also suggests that variety Malayan Tall probably shares common
ancestry with variety Kalok. In contrast, he further observed that variety Pak
Chok could be compared with other talls that have Niu Kafa type character-
istics, for example the talls from Sri Lanka, India, Mozambique, West Africa,
and the Caribbean and Atlantic coasts of America. Rattanapruk (1970) also
stated that the variety Pak Chok found growing along the coast of Indian
Ocean resembles coconut from Sri Lanka. Interestingly, the variety ecotype
Kalok in Perera et al. (1999, 2003) is grouped with varieties San Ramon Tall,
Tagnanan Tall, Bali Tall, Rennell Tall and Malayan Tall in the Southeast
Asia/Pacific group of coconuts. Similarly, the variety Pak Chok is grouped
with those from Sri Lanka, Andaman Islands in the Indian Ocean and
Mozambique.
The grouping of Panama Tall (varieties Panama Manarge and Panama
Aguadulce, both from the Pacific coast of Panama) with Southeast Asian and
Pacific talls is in agreement with Whitehead’s (1976) observation of an eastward
movement of coconuts from Southeast Asia to the Pacific region and subse-
quently from there to the Pacific coast of America. These results are largely in
agreement with the results from ISTR analysis (Rohde et al. 1995), which
grouped Panama Tall with Polynesian varieties/populations of coconuts.
Results of all these studies finally support the conclusion that Cocos nucifera
is divided into two large genetic groups, the Southeast Asia and Pacific group
and the Indo-Atlantic group.
The grouping of all dwarf forms from different geographical regions in a
single cluster within the main South Asia and Pacific group and the Niu Vai
type of coconuts and the loss of allelic richness observed in dwarfs suggest that
dwarfs have a common origin and evolved from the Southeast Asia/Pacific
group of talls in the Southeast Asia/Pacific region. The results of Teulat et al.
(2000) strongly support a common origin of dwarf varieties.
Attempts to investigate the genetic lineages in coconuts using three coconut
specific chloroplast microsatellite primers developed by sequencing and
isolating mononucleotide microsatellite motifs observed in the PCR amplified
chloroplast regions of intergenic spacers between trnT and trnL, trnS and trnT,
and trnC and trnD, on a set of 130 coconut genotypes from all over the world,
failed to show any chloroplast variation, thus indicating a very close ancestral
relationship between coconut groups (Perera 1999, 2002).
12.8.3 Hybridity Testing and Variety Identification
Perera et al. (2004) reported the successful use of microsatellite markers to

uniquely identify seed parents in the Sri Lanka coconut breeding programme
and the resulting hybrids. This is very important for confirming the identity of
varieties and their hybrids in the most likely events of mixing of seed-nut lots,
mislabeling of seeds in nurseries and checking the legitimacy of hybrid
seedlings. Two SSR primers have exhibited the potential for distinguishing
between coconut varieties Sri Lanka Tall, Sri Lanka Green Dwarf and Sri
Lanka Yellow Dwarf used as parents in Sri Lanka. This has led to the unique
capability of conformity and hybridity testing of two commercially grown
coconut hybrids, Sri Lanka Green Dwarf Sri Lanka Tall and Sri Lanka
Yellow Dwarf Sri Lanka Tall. Similarly Bandaranayake et al. (2005) have
developed markers specific to the San Ramon variety for identification of
uncontaminated materials for multiplication in a seed garden.
12.8.4 Somoclonal Variation in Coconut Plants
Application of microsatellite markers to study any possible somaclonal varia-

tion in a limited number of clonal plants of coconut regenerated from various
explants through somatic embryogenesis and their successful field planting in
Sri Lanka have been reported by Fernando et al. (2004). The microsatellite
markers have confirmed the absence of genetic variants among plants within
clones (Fernando et al. 2004).
12.8.5 Linkage Mapping and QTL Identification
Studies on coconut genome mapping have commenced only recently. The first
genome map for coconut was developed for an East African Tall Laguna Tall
F1 population based on ISTR markers (Rohde et al. 1999). This work was
extended with a mapping population developed in the Philippines from a cross
between Malayan Yellow Dwarf Laguna Tall using AFLP, ISTR, RAPD
and ISSR markers. Three hundred and eighty two markers have been placed in
the map resulting in 16 linkage groups and leading to the identification of six
QTLs for early germination (Herran et al. 2000). Genetic correlations have been
established between early germination and early flowering, and early germina-
tion and high yield. Thus, this has become the first report of the opportunity for
marker assisted selection in coconut. Further, QTL for other traits such as leaf
production, girth and height have also been identified for the same mapping
population (Ritter et al. 2000). In addition to this, another mapping population
in Ivory Coast derived from Cameroon Red Dwarfs and Rennell Island Tall has
been used to map 280 markers on 16 linkage groups resulting in several QTLs
12 Coconut 389
related to nut number, number of bunches and traits related to fruit

components (Lebrun et al. 2001; Baudouin et al. 2006).
The size of the mapping population and selection of parents for the
maximum segregation of traits are critical when producing a genome map
of any crop (Bandaranayake and Kearsey 2005). The size of the mapping
population is particularly a crucial issue in coconut because obtaining a
reasonable number of seed nuts from a particular cross is a difficult task as
a result of very low seed production in coconut, i.e. about 100 seed nuts per
palm per year. From a simulation study, Bandaranayake (2006) concluded
that about 400 individuals represent an effective size of a mapping popula-
tion in coconut for a consistent map resolution. However both linkage maps
described in Herran et al. (2000) and Lebrun et al. (2001) were based on the
genotypic and phenotypic scores of less than 65 individuals in their mapping
populations. The size of these mapping populations thus would not be
sufficient for constructing a reliable map because any mapping population
with less than 100 meioses is unlikely to be useful for generating a map
(Young 1994). Furthermore, genetic relationship studies in coconut as
described above suggests that maximum segregation of traits in coconut
can only be obtained by crossing varieties of Southeast Asia and the Pacific
group with those from Indo-Atlantic group. However, all the mapping
populations mentioned were composed of parents belonging to one genetic
group, the Southeast Asia and Pacific group. As a result, about 84% of
DNA fragments generated in the Malayan Yellow Dwarf Laguna Tall
mapping population were non-polymorphic indicating that the two parents
share identical alleles at a large number of loci.
Based on the experience and the information already generated, a large
mapping population has now been created in Sri Lanka comprising 350
individuals arising from a cross between Sri Lanka Red Dwarf (Southeast
Asia and Pacific group) and Sri Lanka Tall (Indo-Atlantic group) to obtain
maximum segregation of traits and sufficient number of meioses to analyze.
Another mapping population, particularly for segregating for tolerance to
Aceria mite, is being constructed in Sri Lanka between tolerant Sri Lanka
Yellow Dwarf and a highly susceptible Sri Lanka Tall palm (Perera 2006).
Generation of new mapping populations including phytoplasma-resistant
breeding material is being focused in Jamaica (unpublished).
12.8.6 Synteny Studies
Efforts are being undertaken to investigate the possible synteny of the oil palm
and coconut genomes as both palm species are diploid and have the same
chromosome number, 2n = 32 (unpublished). Synteny would possibly speed
up research by increasing the marker density on the respective linkage maps
through the exchange of DNA markers.
12.8.7 In Vitro Culture
Coconut tissue culture has a long history dating back to the 1970s. Since then
the problem of cloning coconut has been addressed in a number of research
centers worldwide. Initial attempts were on reversion of floral meristem into
vegetative growth but later on, most efforts were concentrated on somatic
embryogenesis. During the past decade, the in vitro plant regeneration protocol
has been improved, and a limited number of clonal plantlets obtained mainly
from zygotic tissues has been produced in a few laboratories (Chan et al. 1998;
Verdeil et al. 1999; Fernando et al. 2003). However, success is very limited due
to numerous constraints. The regeneration efficiency is far from adequate
though a small number of vegetatively propagated coconut palms have been
established in the field. Due to very poor response of coconut tissues to in vitro
conditions it is classified as one of the most recalcitrant species to regenerate in
vitro (George and Sherrington 1984).
Many farmers still practice selection of their own mother palms for obtaining
seed nuts for planting, thus selection of palms is based on farmer’s long term
observations on the performance of each palm. However, in many countries,
improved seeds are produced by government and/or private sector organiza-
tions and seedlings raised are sold to farmers. When improved seeds need to be
produced on a large scale they are produced in isolated seed gardens. In the case
of hybrid seeds, the mother trees are planted in isolated seed gardens and daily
emasculation of inflorescences is carried out by removing the upper part of each
spikelet that carries male flowers. The remaining female flowers receive pollen
by natural means, from male parents inter-planted at low ratio within the seed
garden, or artificially from pollination labourers visiting each receptive palm,
either by using pole-mounted pollen blowers or by using ladders and hand-
pollinating each female flower with small brushes. When improved seeds are
produced from mass selected high yielding palms or from paired crosses
between selected trees, these are planted in the seed garden and allowed to
naturally inter-pollinate. The second generation seeds obtained this way are
then distributed to farmers for planting. However, production of improved seed
often does not meet the demand from farmers, and hence the balance seed nut
requirement is obtained from superior mother palms selected in high yielding
blocks from high yielding estates. Seed from those trees results from open
pollination.
Acknowledgements Authors thank Dr. R.R.A. Peries of Department of Primary Industries,

Victoria, Australia for his valuable comments, Ms. Mithila Jayasundara of Coconut
Research Institute of Sri Lanka for proof reading and the moderator of the Coconut Time
Line (http://cocos.arecaceae.com) for assistance with citations.
12 Coconut 391
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52, 421–434.
Chapter 13
Olive
Luciana Baldoni and Angjelina Belaj
13.1 Introduction
13.1.1 Overall Importance of the Crop and Production Areas

The olive (Olea europaea L.), grown on over 8 million hectares, is the second
most important oil fruit tree crop worldwide after oil palm and its cultivation is
traditionally concentrated in the Mediterranean area. The total olive oil pro-
duction for the 2006–2007 season was 2,859,500 tons (International Olive Oil
Council (IOOC) data). Southern European countries account for about 74.9%
of the world production, with Spain being the main producer (38.7%), followed
by Italy (21%) and Greece (12.9%). Other important olive oil producers are
Turkey, Tunisia and Syria (17.1%) as well as Jordan, Morocco and Algeria.
Seventy percent of the olive oil produced globally is consumed in the Med-
iterranean area, but its demand is rapidly increasing in other countries. For
example, the consumption of olive oil in the USA grew from 88,000 tons in 1990
to 251,000 tons in 2007, in Japan from 4,000 to 31,000, and in Australia from
13,500 to 40,000 tons (IOOC data).
13.1.2 Major Problems of Olive Cultivation
The income from olive production is quite low due to the low mean production
per unit area of about 2 t/ha of olives, the low content of oil rarely exceeding
24% of fresh weight (depending on variety, agro-climatic conditions and
extraction method) and the high costs of cultivation. Fruit production starts
3–5 years after planting, and olive orchards can survive almost indefinitely due
to the longevity of the species. However, alternate bearing and the costs of
cultivating old trees generally force the reduction of the lifetime of olive orch-
ards to no more than 50–60 years. In order to minimize the costs of production,
L. Baldoni (*)
CNR, Istituto di Genetica Vegetale, Perugia, Italy
e-mail: luciana.baldoni@igv.cnr.it

398 L. Baldoni and A. Belaj
new models of cultivation have been developed based on the complete mechan-
ization of harvesting and pruning, the most time-consuming steps in oil
production.
The olive is a long-living evergreen tree that grows up to 15 m at maturity. Its
life span is typically longer than 500 years, and trees older than 2,000 years are
still under cultivation in numerous regions (Fig. 13.1). Flowers are generally
hermaphroditic and wind pollinated, and most cultivars are self-incompatible.
The olive fruit is a drupe (Fig. 13.2) and, in contrast to other oil crops,
the oil accumulates in the mesocarp, and only 3–4% of the oil is derived
Fig. 13.1 Ancient olive still under cultivation in Apulia (Italy). Olive trees may survive
for thousands of years and many of them can be found along the entire Mediterranean
area of cultivation. Plants like this represent the most ancient cultivated living plants of the
world
13 Olive 399
Fig. 13.2 Green olive drupes; at ripening the colour will turn to reddish or black
from the seed. The best olive oils are extracted from olives harvested at the
green-turning-to-black stage of ripeness; they have to be immediately milled to
minimize oxidation and enzymatic reactions.
13.1.3 Types of Olive Oil and Characteristics
Virgin olive oil is mechanically extracted by pressing or centrifuging crushed

fruits without any chemical treatment. Main component of olive oil triglycer-
ides is the mono-unsaturated oleic acid (18:1), which represents 57–80% of total
fatty acids, followed by linoleic (18:2) (7–19%), linolenic (18:3) (0.6–0.8%),
palmitic (16:0) and stearic (18:0) acids (Salas et al. 2000).
Due to the mechanical processing, virgin olive oil represents a fresh-squeezed
fruit juice containing, other than triglycerides, a vast range of microconstituents
(more than 230 compounds) including phenolics, tocopherols, aliphatic and
triterpenic alcohols, sterols (and their precursor squalene), hydrocarbons and
volatile compounds, representing up to 20% of the fresh olives and about 2% of
virgin olive oil (Obied et al. 2008). The phenolic compounds of olive and virgin
olive oil show a peculiar composition that can not be found in any other
vegetable oil. They include different types of hydrophilic phenols, such as
secoiridoids, lignans, flavonoids, phenolic alcohols, and phenolic acids. Secoir-
idoids are the most important class of phenolics and they arise from simple
structures, like tyrosol and hydroxytyrosol, to quantitatively more important,
conjugated forms, like oleuropein and ligstroside (Servili et al. 2004). The pro-
tective activity of olive oil on the prevention of a variety of tumors and important
chronic and degenerative diseases may be credited to these minor constituents
unique to virgin olive oil. There is extensive background demonstrating the
effectiveness of olive oil and some of its compounds on human health.
Oleuropein, exclusively present in olive and in a few other related taxa, is a
non-toxic secoiridoid known to exhibit several biological properties, many of
which may result from their antioxidant and free radical scavenger activity. A
number of observations elevate oleuropein from a non-toxic antioxidant into a
potent anti-tumor agent with direct effects against tumor cells (Hamdi and
Castellon 2005; Giamarellos-Bourboulis et al. 2006). Beauchamp et al. (2005)
have recently identified oleocanthal (deacetoxy ligstroside aglycon), a com-
pound present in newly pressed extra-virgin olive oils that was shown to be a
3–4 times more potent inhibitor of Cox-1 and Cox-2 than ibuprofen, a potent
modulator of inflammation and analgesia (Galli 2006). Squalene is a triterpene
containing six isoprene units and representing a key intermediate in the biosyn-
thetic pathway to steroids in plants and animals. It has been demonstrated to
protect against skin cancer (Newmark 1999). Minor compounds are also respon-
sible for the organoleptic qualities, taste and flavor of extra virgin olive oils,
which may distinguish oils originating from different regions.
As defined by the European Union Council Reg. (EC) 1513/2001, there are
additional categories of olive oils: Refined oil is obtained from low quality virgin
olive oil after chemical and physical refining treatments, which lead to removal
of contaminants or degraded compounds. Olive oil consists of a blend of refined
and virgin olive oil. Finally, olive-pomace oil is obtained by treating olive
pomace with solvents.
Within the category of virgin olive oil the extra virgin is far more valuable than
most other vegetable oils, due to the low percentage of free fatty acidity (<0.8%)
and the superior taste, but its production is costly and time consuming. For this
reason adulteration is very common, mainly represented by blending premium
extra virgin olive oil with seed oils or cheap olive oil. In order to reduce frauds,
valorize excellence and defend the origin of best products, a number of regula-
tions and standards have been defined by the International Olive Oil Council and
by the Codex Alimentarius Commission. Moreover, the European Union has
established the option of a protected designation of origin (EU Commission Reg.
(EC) 1898/2006) for olive oils of important regional and traditional origins.
The quality of extra virgin olive oils strongly depends on the cultivar of
origin, which affects the content of specific polyphenols and aromatic com-
pounds controlling taste and flavour of the oil. Agro-climatic factors, harvest-
ing time and oil extraction technologies may have a negative effect on the olive
oil quality, altering the fatty acid and phenolic composition.
DNA fingerprinting is a powerful aid for the identification of olive oil
provenance, and fingerprinting methods have been applied to trace the varietal
origin of batches of olive oil (Muzzalupo et al. 2007; Busconi et al. 2003; Breton
et al. 2004; Pasqualone et al. 2004; Testolin and Lain 2005).
13 Olive 401
13.1.4 Ancient and Recent History of Olive Cultivation
Olives have been extensively cultivated along the Mediterranean basin since
3000 BC and have had an enormous impact on the economy, history, culture,
and environment of the area. Ancient Greeks and Romans considered olive oil a
sacred substance and used it as food, medicine, soap, fuel for lamps and as base
for perfumes. There are a great number of archaeological remains showing the
cultivation, extraction, commerce, and consumption of olive oil from the main
civilizations along the Mediterranean region.
The first evidence for olive cultivation is seen during the Minoan age, when
olives were cultivated on the island of Crete (3000–1500 BC). They were then
cultivated by the Egyptians (2000 BC) and grown in an almost specialized form.
In 1000 BC olive started to be cultivated in Palestine and, between the ninth and
eighth centuries BC, olive growing was seen in Greece and in North African
coasts surrounding the Mediterranean Sea, where it was introduced by the
Phoenicians. Thanks to the Phoenician and Greek shipping routes, olive trees
reached the coasts of Sicily and Spain, where they were widely diffused in the
fifth century BC. Between the sixth and fourth century BC their cultivation was
established in many regions of Italy and Spain by the Romans. By the first
century AD, olives were a cash crop for the Romans, who imported oil from the
most remote colonies of the empire, mainly Spain and North Africa. Olive
cultivation declined during the Medieval age but increased again after the 15th
century, up to the present extension of cultivated area in the Mediterranean
(Mastrangelo 1982).
After several unsuccessful attempts to introduce olive cultivation in Central
and North America during the 18th century, olive production has recently
started in new countries such as Argentina, Chile, Mexico, USA (California),
New Zealand, Australia, and South Africa, that now represent 1.4% of world
olive production.
The poor historical documentation on cultivar pedigrees and the fragmented

information available on olive paleobotany have failed to provide definitive
conclusions on the origin of the cultivated olive, but numerous hypotheses are
currently under evaluation. Palynological, anthropological, and archeological
evidence (Watts et al. 1996; Carrion and Dupré 1996) have demonstrated the
presence of sporadic forms of olive during the last glaciation (18000 BC) in the
western and eastern Mediterranean regions.
Oleasters have been directly exploited for oil extraction since 4500–4300 BC as
evidenced by archaeological and paleobotanical findings from Spain (Terral et al.
2004; Rodrı́guez-Ariza and Montes Moya 2005). Some studies on olive domes-
tication have asserted that cultivars moved westward with human migrations
(Besnard et al. 2001a; Belaj et al. 2002). Other recent evidence has pointed out
that the domestication process started simultaneously at both ends of the
Mediterranean (Lumaret et al. 2004; Breton et al. 2006). Analyses of archae-
ological charcoal and olive stones have effectively dated domestication to the
end of the Bronze Age in the north-western Mediterranean area (Terral 2000;
Terral et al. 2004; Rodrı́guez-Ariza and Montes Moya 2005).
The first cultivars were probably selected from trees bearing large fruits and/or
high oil content and were vegetatively propagated, either via cutting or grafting
onto indigenous oleasters. However, considering the long life of olive plants, there
has been relatively little selection and probably only a few generations separate the
presently cultivated forms from their progenitors (Liphschitz et al. 1991).
Taking into account recent results obtained by molecular analysis of sets of
wild olives and cultivars from different Mediterranean regions, it has been
assumed that olive trees have undergone different selection/domestication pro-
cesses in different regions. The contribution of local wild plants to the develop-
ment of varieties has been demonstrated only in restricted areas where wild
olives, very ancient cultivated trees, and local varieties shared a large portion of
variability. In other areas of the western Mediterranean, in contrast, the clear
distinction between oleasters and cultivars has confirmed that local cultivars
did not develop from local oleasters but were introduced from abroad and
propagated by grafting onto local oleasters (Baldoni et al. 2006).
13.3.1 Cultivated Olive Germplasm

Olive germplasm has not suffered significant genetic erosion, maintaining
almost intact its entire variability, given that turnover with new genotypes has
not occurred, and that the species has great longevity and a good capacity to
survive without cultivation. Thus, more then 1,200 varieties are still under
cultivation, 79 national and international collections hold about 4,200 geno-
types and more than 5,300 cultivar names are recognized (Bartolini et al. 1998).
Over two thirds of cultivars are present in the southern European countries (538
in Italy, 183 in Spain, 88 in France, 52 in Greece and 45 in Turkey) (Bartolini
et al. 1998), and many other local varieties and ecotypes contribute to the
richness of the olive germplasm. Olive represents, therefore, an unusual case
among horticultural crops and its germplasm could constitute a particularly
rich source of variability to be used directly or for future breeding.
Olive cultivars can be considered as varieties of unknown origin, most of
them originating from empirical selections made by growers from naturally
cross-bred genotypes over many centuries and propagated by cutting or graft-
ing. Evidence for multilocal selection of most cultivars has been repeatedly
demonstrated (Besnard et al. 2001b; Rotondi et al. 2003).
13 Olive 403
The genetic diversity of cultivated populations shows a complex patchy

pattern (Baldoni et al. 2006; Owen et al. 2005). Few cultivars are dispersed
over widespread areas, whereas the majority of varieties are highly localized.
Cultivars may either have a non-autochthonous origin or have been derived by
selection from local oleasters (Lumaret et al. 2004; Breton et al. 2006).
Up to now, identification of each variety has been delayed by confusion
about the names given to each genotype, the low intervarietal genetic distances,
the intra-cultivar variability and the putative presence of asymptomatic viruses
that may affect the plant phenotype.
13.3.2 Cultivar Classification

The geographical distribution of olive cultivars and their economical importance
are considered the main criteria for their classification. According to this system,
Barranco and Rallo (2000) divided olive cultivars into four categories: main,
secondary, dispersed and local. Main cultivars are those which account for either
a large portion of the acreage or predominate in one or more olive districts.
Secondary cultivars are not predominant in any district but form the basic cultivars
of some orchards. Dispersed and local cultivars are isolated trees in various or
single districts, respectively. The agronomical performance of main cultivars has
been widely evaluated, while for most of the others information is unavailable.
13.3.3 Identification of Olive Cultivars

Until recently, the variability of O. europaea germplasm has been reported with
respect to morphological descriptors. Understanding the amount and distribu-
tion of genetic variability among cultivars by means of molecular markers has
been the main goal for most of research on olive trees.
Various types of molecular markers have been widely applied over the last
decade, particularly in investigations aiming at studying the variability of olive
cultivars and at developing tools to determine their origin and detecting frauds on
olive oil varietal composition (Hess et al. 2000; Rallo et al. 2000; Sefc et al. 2000;
Guerin et al. 2002; Owen et al. 2005; Montemurro et al. 2005; Vargas and Kadereit
2001; Carriero et al. 2002; Cipriani et al. 2002; Bandelj et al. 2002; Sarri et al. 2006).
Isozymes were the first molecular markers used (Trujillo et al. 1995). Since
then, cultivar identification has been based on DNA markers such as RAPDs
using different protocols (Mekuria et al. 1999; Belaj et al. 2002; Gemas et al.
2000; Sanz-Cortés et al. 2001; Gonzalo-Claros et al. 2000). AFLP data are
available on a wide number of cultivars (Angiolillo et al. 1999; Baldoni et al.
2000; Sanz-Cortés et al. 2003; Sensi et al. 2003; Owen et al. 2005), ISSRs
(Pasqualone et al. 2001; Vargas and Kadereit 2001) have also been used and
SCAR markers have been developed from RAPDs (Hernandez et al. 2001).
To date, very few SNP markers have been identified in olive. Based on the
sequence of candidate genes and their frequency along coding sequences, it has
been estimated that there is one SNP in every 190 base pairs (Reale et al. 2006;
Consolandi et al. 2007).
For cultivar characterization simple sequence repeats (SSRs) have become
the most popular kind of marker (Sefc et al. 2000; Rallo et al. 2000; Carriero
et al. 2002; Cipriani et al. 2002; De la Rosa et al. 2002; Dı́az et al. 2006a; Sabino
Gil et al. 2006), and it has been demonstrated that only three SSR markers are
able to distinguish more than a hundred olive genotypes (Sarri et al. 2006). In a
recent paper a set of the most effective SSR markers has been selected and
proposed for varietal characterization (Baldoni et al. 2009).
DNA markers used to identify olive cultivars have also been applied for
DNA tracking of olive oils to test their varietal composition (Breton et al. 2004;
Pasqualone et al. 2004; Pafundo et al. 2005; Testolin and Lain 2005; Doveri
et al. 2006; Muzzalupo et al. 2007; Consolandi et al. 2008).
13.4.1 Taxonomy and Distribution of Olea europaea
The genus Olea belongs to the Oleaceae family, sub-family Oleideae. The genus
includes two sub-genera: Olea and Paniculatae, the former is divided into
sections Olea and Ligustroides. According to recent revisions of Olea europaea
taxonomy, the species includes six sub-species based on morphology and geo-
graphical distribution (Green and Wickens 1989; Green 2002):
– subsp. europaea, represented by two botanical varieties: cultivated olive
(var. europaea) and wild olive (var. sylvestris), both present throughout
the whole Mediterranean basin;
– subsp. cuspidata, distributed along south Asia, on the Arabian peninsula,
and throughout east and south Africa;
– subsp. laperrinei, restricted to the Sahara region;
– subsp. maroccana, present in Morocco;
– subsp. cerasiformis, typical of Madeira island;
– subsp. guanchica, restricted to the Canary Islands.
Wild and cultivated olives are diploid (2n = 2x = 46), predominantly allo-
gamous and their genome size is about 1,800 Mb (Loureiro et al. 2007; Besnard
et al. 2008).
13.4.2 Natural Diversity of Olive

The geographic distribution of variability within the Olea genus and the
genetic relationships among the wild (oleasters) and cultivated olives have
been studied using various molecular methods (Angiolillo et al. 1999;
13 Olive 405
Besnard et al. 2002a,b; Lumaret et al. 2004; Baldoni et al. 2006; Breton
et al. 2006; Belaj et al. 2007; Rubio de Casas et al. 2006).
The wild relatives of cultivated species exhibit traits such as biotic stress
resistances, adaptation to extreme environmental conditions, plant vigour and
architecture, which could be utilized in olive breeding. Model plant genetic
systems and the molecular genetic resources that are currently available are
greatly enhancing our ability to identify adaptive or stress-responsive genetic
determinants. But natural diversity is still an underexploited sustainable
resource for olive that could enrich the genetic basis of cultivated plants with
novel alleles to improve both productivity and adaptation.
The potential value of wild olive trees and related species as a source of
agronomically interesting traits has never been evaluated, thus severely
restricting the ability of breeders to develop new genotypes by introgres-
sion of superior alleles into cultivated varieties. Oleasters growing under
the drought-salt-heat complex conditions and ultra-millennially aged olive
trees surviving in adverse environments have only occasionally been sub-
mitted to phenotypic evaluation, and there is a lack of serious prospecting
surveys to characterize wild olive populations (Mulas et al. 2004; Belaj
et al. 2007).
Up to now, most work has concentrated on evaluating the distribution of
variability between cultivated and wild olives and on establishing the genetic
relationships among the different O. europaea subspecies that are distributed
beyond the Mediterranean area.
13.4.3 Wild Olives
Wild olive (Olea europaea subsp. europaea var. sylvestris), also known as
oleaster, has colonized diverse environments along the Mediterranean basin,
characterised by semi-arid climatic conditions with different altitudes, vegeta-
tive communities and soils, including those with extreme levels of drought, low
temperatures and salinity (Baldoni et al. 2006). Wild plants occur in the same
areas as domesticated olive, in the maquis and in uncultivated sites, and show
some morphological differences from cultivars, such as a bushy plant shape and
small fruit size (Terral and Arnold-Simard 1996).
The contribution of oleasters to the evolution of cultivated olive is still ques-
tionable, and a widely debated problem relates to the distinction between real
oleasters and feral plants derived from the natural dissemination of cultivars. In
fact, both forms occur in the same ecological sites and their appearance is very
similar. Different criteria have been proposed to clearly distinguish the two forms
based on geo-ecological parameters (Lumaret et al. 2004) or on molecular
markers (Baldoni et al. 2006; Breton et al. 2006). It is believed that oleasters
have originated in the eastern Mediterranean, and that wild olives present in the
western Mediterranean basin could be feral (Besnard et al. 2002b).
In previous studies performed through the analysis of chloroplast, mito-

chondrial, and nuclear DNA polymorphisms, it has been shown that eastern
and western Mediterranean oleaster populations are strongly differentiated
from one another (Besnard et al. 2001b, 2002b; Lumaret et al. 2004).
It has been hypothesized that humans could have brought eastern-specific
chlorotypes to the west, probably bringing plant material (or olive fruits) from
the eastern to western Mediterranean (Besnard et al. 2002b). The linkage
disequilibrium between widely-spread chlorotypes and nuclear markers char-
acteristic to eastern oleasters can be explained by the common origin of these
polymorphisms in a wild population from the east. These results support the
hypothesis that western oleasters could be feral forms as a result of an eastern
introduction and a gene flow from olive groves towards wild populations.
Studies carried out by the use of allozyme markers (Lumaret et al. 2004) have
pointed out the genetic evidence that genuine oleasters still survive locally in the
west Mediterranean, as shown by west-specific alleles found in wild olives
collected in forests potentially containing genuinely wild forms according
to environmental, historical, and demographic criteria. Western popula-
tions are more closely related to the wild populations of the Canary
Islands. Populations of wild olive seem to be restricted to a few isolated
areas of the native Mediterranean forests, where pollen and stones may be
wind/bird-distributed.
Other studies, performed on oleaster populations of restricted areas on both
sides of the Mediterranean pointed out significant differences between east and
west areas (Rubio de Casas et al. 2006). The highest genetic diversity was found
at the extreme western side of Mediterranean and in the Balearic islands.
Additionally, long-lasting isolation of the northern populations of the Iberian
Peninsula appears to be responsible for a significant divergence. Gene flow
estimates demonstrated that genetic material seems to be exchanged frequently
among populations in accordance with the predominant outcrossing between
wild and cultivated individuals. Birds eating olives (Rey and Alcántara 2000)
may enable long distance dispersal and make exchange of migrants common
even between distant regions. Pollen circulation can also occur over long dis-
tances, enhancing lineage admixture.
The cline of genetic diversity revealed by chloroplast and SSR markers was
explained by oleaster re-colonization of the Mediterranean basin from refugees
after the last glacial event, located in both eastern and western regions (Breton
et al. 2006). Based on different population analysis methods, it has been shown
that oleasters are equally present in the eastern and the western Mediterranean,
and that they are native and not derived from cultivars. It is also likely that gene
flow has occurred in oleasters mediated by cultivars spread by human migration
or through trade and animals.
The evaluation of olive differentiation at a microscale regional level has
shown more complex results (Baldoni et al. 2006). Levels of interpopulation
genetic variance between wild olive populations present in distant islands and
highly differentiated from mainland oleasters, which only partially represent
13 Olive 407
real wild olives, were very low, even if the populations were clearly distinguish-
able from those of other areas. Other wild olives represent feral plants, spread in
the same uncultivated areas where real oleasters still survive. The low level of
differentiation among wild plants of distant isolated regions is difficult to
explain for natural populations. Factors that may be considered include a
common ancestral genetic pool, a lack of differential selective pressure and a
reduced number of divergent generations between the different populations,
likely representing refugial relics of the same population.
13.4.4 Related Subspecies

Analyses performed on rDNA, cpDNA, and mtDNA polymorphisms have
characterized three distinct main clades: O. europaea – O. laperrinei –
O. maroccana – O. cerasiformis (¼ europaea phylum) of the Mediterranean
region, Sahara and northwest Africa, O. cuspidata – O. chrysophylla (¼ cuspi-
data phylum) of Asia, and O. africana (¼ africana phylum) for east and south
Africa (Besnard and Bervillé 2002; Besnard et al. 2002a,b).
AFLP analyses showed that subsp. laperrinei and maroccana, from north-
west Africa, showed a high similarity with the Mediterranean cultivated and
wild olive and a clear distinction of the Australian taxa from those of east Africa
and Asia, which clustered together (Angiolillo et al. 1999).
The phylogenetic relationships between the Olea europaea subspecies and
other related taxa were assessed by nucleotide variation in non-coding cpDNA
regions and by cpDNA RFLPs, distinguishing four groups: the taxa of north-
west Africa and Mediterranean region (including the cultivated olive), the
O. europaea forms from southeast Africa, those from Asia, and finally Olea
capensis and O. lancea, both belonging to a distinct subgenus Ligustroides
(Baldoni et al. 2002; Lumaret et al. 2000).
Two tandemly repeated sequences isolated by Katsiotis et al. (1998) localized
on the chromosomes by in situ hybridization are present in oleasters and in subsp.
chrysophylla and africana, but are absent in other genera of the Oleaceae family.
Despite the pressure to improve productivity and agronomic performance of

olive cultivars and in spite of the economic importance of the crop, there have
been few efforts to produce new olive cultivars.
Exploration of phenotypic variability in agronomic characters has led to the
identification of valuable clones within numerous olive cultivars of various
Mediterranean countries (Suárez et al. 1990; Lavee et al. 1995; Bartolini et al.
2002; Grati-Kammoun et al. 2002). However, in spite of the significant efforts
made towards clonal selection, very few clones have shown outstanding per-
formance (Loussert and Berrichi 1995; Tous et al. 1998).
Very few studies have addressed the selection of clonal rootstocks. Prelimin-
ary work addressed the influence of rootstocks on scion performance. Clonal
rootstocks have been selected with high rooting ability and the capacity to
control scion vigour (Baldoni and Fontanazza 1990). Other selected rootstocks
can control scion vigour and resistance to frost injury (Pannelli et al. 2002). The
use of cvs. Souri, Muhasan and Barnea as rootstocks under dry conditions did
not show any significant effect on tree vigour, shape and fruit production after
ten years from planting (Lavee and Schachtel 1999).
Similarly to clonal selection, the use of induced mutagenesis has not been
encouraging, and so far has succeeded in producing of only a compact mutant
of the cv. Ascolana Tenera (Roselli and Donini 1982).
The evaluation of minor local cultivars has recently been exploited to iden-
tify individuals highly adapted to extreme environmental conditions (Pannelli
et al. 2003; Rotondi et al. 2003).
The long generation time of olives has severely hindered both classical
breeding and genetic studies (León et al. 2004a; Santos-Antunes et al. 2005).
But now the development of new protocols to force seedling growth has made it
possible to greatly reduce the length of the juvenile phase, even if the evaluation
of the agronomic performance of mature plants still requires at least five years
of experimentation (Santos-Antunes et al. 2005). Furthermore, the genetic
control of major traits under selection is still unknown (De la Rosa et al.
2003). Tree vigour, leaf size, and fruit shape seem to be controlled by major
genes showing dominance (Bellini et al. 2002a), while the inheritance of other
characters such as fruit size, flowering intensity, fruit set, ripening time, and
yield remains uncertain (Bellini et al. 2002a; Parlati et al. 1994).
In spite of these drawbacks, various classical breeding programs, performed
by intervarietal crossing have been reported from different countries such as
Turkey (Arsel and Cirik 1994), Morocco, Spain (Rallo 1995; León et al. 2004a,
2007), Tunisia (Trigui 1996), Israel (Lavee et al. 1999, 2003), Greece (Pritsa
et al. 2003), Italy (Fontanazza et al. 1998; Bellini et al. 2002b) and Iran. In
Australia, the University of Adelaide has recently established a breeding pro-
gram to select for quality oil production in feral olive populations derived from
natural dissemination of cultivars previously introduced in Australia and well
adapted to that environment (Sedgley 2004). In any case, at present, the
procedures of selection are still in progress and very few new genotypes have
arisen from these breeding programmes.
Maalot, a new cultivar resistant to Spilocaea oleagina, has been selected from a
selfed F1 progeny of a semi resistant seedling probably of cv. Chemlali (Lavee
et al. 1999). From seedling populations obtained by unknown parents, two other
cultivars were selected, ‘Barnea’ with vigorous and upright growth and ‘Kadesh’
as a table olive (Lavee 1978; Lavee et al. 1986). The new cultivar ‘Askal’, a hybrid
from the cross ‘Barnea’ ‘Manzanillo’, was selected for its adaptation and good
performance in high-density olive orchards (Lavee et al. 2003).
Three new olive cultivars (‘Arno’, ‘Tevere’ and ‘Basento’) were released from
the progeny of the cross ‘Picholine Manzanillo’ (Bellini et al. 2002b) and their
13 Olive 409
performance is still under evaluation. The new cultivar ‘Fs 17’ was selected
among seedlings obtained from free pollination of the Italian olive cultivar
‘Frantoio’ (Fontanazza et al. 1998).
Very recently, the new cultivar ‘Chiquitita’, derived from a cross between
‘Picual’ and ‘Arbequina’, was obtained in a Spanish cross-breeding program.
This new cultivar is characterized by early bearing, high oil content and high
yield efficiency, while its low vigour, compact canopy and pendulous branches
make it very suitable for high density orchards (Rallo et al. 2008).
In the same olive breeding program, preliminary results obtained from a com-
parative trial of the first 15 selections (León et al. 2007) indicated that some early
bearing genotypes could be released as new cultivars. Additionally, some selections
have shown a low vigour and could be kept for high-density hedgerow orchards.
The primary goals in olive breeding are directed towards overcoming current
limiting factors for production. These include: shortening the unproductive
period, increasing fruit number and size, increasing oil content and quality
(fatty acid composition, phenol content, etc.), reduction of alternate bearing,
dwarfing or modifying tree architecture to facilitate mechanical pruning and
harvesting, improving resistance to pests, in particular olive fruit fly, Bactrocera
oleae, and diseases such as leaf peacock spot, caused by Spilocaea oleagina,
Verticillium wilt, Verticillium dahliae and olive knot, Pseudomonas savastanoi.
Other important objectives are to improve cold tolerance to allow cultivation in
colder areas and to promote self-fertility in order to reduce reliance on
pollinators.
Tree architecture and vigour are particularly important because the height of
the trees prevents mechanical harvesting and pruning, thereby increasing the
costs of cultivation.
Although olive is considered a species adapted to semi-arid climates, its
productivity is strongly reduced under dry conditions, and thus there is great
interest in developing new drought-tolerant cultivars as well as those that can
thrive on saline and heavy soils.
Rootstock selection is focused on the ability to control scion vigour, to
improve the resistance to pathogens, mainly Verticillium, and to abiotic stresses,
namely water stress.
13.7.1 Classical Breeding

Main steps for olive breeding are: (i) establishing the inventory of the existent
varieties and determining their agronomic value, and (ii) breeding by hybridi-
sation and selection. The first point has received particular attention and is
under way in various Mediterranean countries. The second step has been
initiated in Spain and in a few other countries.
13.7.2 Clonal Selection
Clonal selection aims at uncovering valuable genotypes within a variety. Most

selection programs in olive have so far relied on clonal selection and are based
on the assumption that natural mutations generating any positive alteration in
traits of agronomic interest may occur in long-living plants, in which they may
be maintained by vegetative propagation (Rallo 1995; Belaj et al. 2004). Pro-
specting surveys to identify outstanding trees within a variety, either for agro-
nomical or technological characters, are the first steps for clonal selection.
Vegetatively propagated progenies of these individuals are then tested in com-
parative trials. Individuals performing better than standard samples of the
corresponding cultivar for specific characteristics are selected and their vegeta-
tively propagated clones represent the new clonal selections. The selected
individuals most commonly retain the original cultivar name and acquire an
identifying clone code. Most of the reported works on clonal selection in olive
have followed this methodology, but most of them have stopped at the first step.
This method of selection has demonstrated a low efficiency, and only a few
selected clones have gained commercial relevance and have been propagated by
the nursery industry (Loussert and Berrichi 1995; Tous et al. 1998).
13.7.3 Sanitary Selection
Systemic pathogens, such as viruses and phytoplasms, are sources of variation in

vegetatively propagated plants. The application of molecular diagnostic techni-
ques (such as RT-PCR) has allowed to detect the presence of different viruses
(Faggioli et al. 2005) which may be symptomless but affecting plant morphology.
Little information on the incidence of these diseases on agronomic characteristics
is available and has given contradictory results (Clara et al. 1997; Martelli 1998;
Bartolini et al. 1998). For that reason in the last years the sanitary selection, i.e.
original plant material free from systemic pathogens, has become a technique of
olive selection (Bottalico et al. 2004).
13.7.4 Breeding by Intervarietal Crossing
The adequate choice of parents is of great importance for the achievement of the
objectives of a breeding program. Thus, a detailed knowledge of cultivars’
identity and of their agronomic performance as well as the amount and dis-
tribution of their genetic variability is crucial to broaden the genetic base of new
cultivars (Belaj et al. 2004; León et al. 2004a).
In olive breeding, the length of the juvenile period has traditionally been one
of the main drawbacks. Under normal conditions olive seedlings begin to set
fruits 15–20 years after germination (Santos-Antunes et al. 2005). This may
13 Olive 411
explain the various attempts by olive breeders to study the juvenile period and
to develop protocols to shorten it (Lavee et al. 1996; Santos-Antunes et al. 2005;
De la Rosa et al. 2006).
The protocols under use to perform intervarietal crosses in the olive consist of
adding pollen of the paternal variety to bagged branches of the maternal parent,
chosen among self-sterile cultivars (Lavee 1990; Fontanazza and Baldoni 1990;
De la Rosa et al. 2004). Fruits are collected at ripening and, after removing the
endocarp, seeds are germinated under controlled temperature and humidity.
Plantlets are grown in a greenhouse under continuous light until they reach a
minimum height, then they are moved to the field where they undergo the
procedures of agronomic evaluation and selection of outstanding genotypes.
After the pre-selection phase, plants are vegetatively propagated and compared
in experimental trials for the final selection (Lavee et al. 1999; León et al. 2004b;
Santos-Antunes et al. 2005; León et al. 2007).
An alternative way to overcome the problems of the long juvenile period is the
selection of early bearing genotypes (Pritsa et al. 2003; De la Rosa et al. 2006).
The relationships between seedling phenotypes and their agronomic behaviour at
the mature phase are also very important. The initial results of a comparative trial
of some genotypes under selection indicate that seedlings with a short juvenile
period also show a short unproductive period when vegetatively propagated
(León et al. 2007). It has also been demonstrated that there is a strong correlation
between the resistance to Spilocaea oleagina of seedlings and that of adult plants
(De la Rosa et al. 2006; Lavee et al. 1999; León et al. 2007).
The first evaluations of olive progenies have shown wide ranges of variation
for all evaluated characteristics (Lavee et al. 1999; León et al. 2004a,b). Sig-
nificant correlations have been observed among many traits. The most relevant
correlations were found between oil content and oleic acid concentration, which
was negatively correlated with palmitic, palmitoleic and linoleic acid percen-
tages (León et al. 2004b).
Up to now, crossing programs have been performed only between cultivars,
but the use of wild olives in future crosses should introduce useful variability, as
wild genotypes may contain characteristics rare or absent in cultivated olive
germplasm. So far, only one case of an interspecific cross has been reported
using Olea chrysophylla Lam (Lavee 1990).
At the moment, olive breeding programs using intervarietal or interspecific
crosses are mainly carried out in Spain, Israel and Australia.
13.7.5 Marker Assisted Breeding
The very preliminary works performed on olive genomics are far from producing
effective results toward the selection of new cultivars by the use of molecular
tools. For that reason and considering the lack of knowledge on the real useful
variability already present in the cultivated and wild olive germplasm, attention
has been focused in the last ten years on the evaluation of such germplasm.
As far as the use of molecular markers in olive breeding programs, a SCAR

marker was proved to be linked to leaf peacock spot tolerance, as reported by
Mekuria et al. (2001). Recently SSR markers have proved to be useful for
paternity testing in olive progenies (De la Rosa et al. 2004; Dı́az et al. 2006a;
Mookerjee et al. 2005) as well as for the study of parental cross compatibility
(Dı́az et al. 2006b). These studies have evidenced the high frequency of con-
tamination with undesirable pollen in seedlings (Santos-Antunes et al. 2005;
León et al. 2004b). SSR markers have also proved to be useful for unequivocally
identifying selections from a breeding program (Dı́az et al. 2007).
One of the major contributions of molecular markers in breeding is the con-
struction of genetic maps and the detection of QTLs. The first mapping population
in olive consisted of a progeny derived from the cross between two highly hetero-
zygous cultivars, Leccino and Dolce Agogia. Dolce Agogia is resistant to the most
important olive pathogens such as Spilocaea oleagina and Verticillium dahliae
(Bartolini et al. 1998; Gonzales-Lamothe et al. 2002), whereas Leccino is suscep-
tible or medium tolerant to these biotic stresses. The linkage map was based on
dominant (RAPDs and AFLPs) along with a small number of codominant
markers (RFLPs and SSRs, De la Rosa et al. 2003). The Leccino map covered
2,765 cM and included 249 markers, falling into 22 major and 17 minor linkage
groups (the latter each involving less than four markers). The Dolce Agogia map
was of similar length (2,445 cM) and included 236 markers arranged in 27 major
and three minor linkage groups. Besides, a candidate gene for stearoyl-ACP
desaturase, which is a key enzyme for the conversion of 18:0 stearic acid to 18:1
oleic acid, the main component of olive oil was mapped on a linkage group of cv.
Leccino (De la Rosa et al. 2003). At present, with the aim to construct a reference
linkage map, this first map is being completed by a wider use of codominant
markers such as SSRs and SNPs.
A second linkage map was constructed by Wu et al. (2004) based on RAPDs,
SCARs and SSRs, exploiting the progeny of a cross between the cultivars
Frantoio and Kalamata. The greater use of codominant markers allowed the
integration of the two parental maps to generate 15 linkage groups covering
101 loci and 879 cM with a mean inter-marker distance of 10.2 cM.
At present, no further olive genetic mapping data is available, no QTLs have
been detected, and neither is there any detailed analysis known on genome
organization.
The very long generation time of olive has delayed the recovery of superior olive
cultivars through conventional breeding. Genetic transformation represents a
powerful alternative technique for accelerating the development of superior
cultivars. For the introduction of genes controlling specific traits via transge-
netics, many studies have been performed in the 1980s through 2000 in order to
develop the biotechnological tools necessary to transform and regenerate olive
13 Olive 413
plants. But the restrictions from the European legislation and the public con-
cern about the use of genetically modified plants, especially for traditional
products such as olive oil, have dramatically decreased investigation on such
topic during the recent years.
Here, the goals and main achievements obtained on the different aspects of
olive genetic transformation will be summarized. Most importantly, olive
transformation research has to address the identification and evaluation of
genes and specific promoters for useful traits and the development of efficient
protocols for regeneration from cell and tissue cultures of elite cultivars. Many
potentially useful genes have been isolated from different species which could be
introduced into olive separately or in combination. However, genetic transfor-
mation also requires the development of genotype-independent procedures
based on the transformation of meristematic cells with high regeneration
potential and/or the use of regeneration-promoting genes.
13.8.1 Organogenesis and Regeneration

Organogenesis represents a first step to somatic embryogenesis, as adventitious
buds developing from explants of micropropagated plantlets may generate
embryogenic cultures.
Shoot organogenesis and complete plants from mature phase explants of
important cultivars have been achieved by Mencuccini and Rugini (1993), and
thereafter applied routinely with minor modifications on the media and regen-
eration conditions. New rooting has been induced by inoculating the basal part
of in vitro microcuttings with Agrobacterium rhizogenes. The application of
putrescine increased rooting rate and basal callus formation (Rugini 1992).
Significant progress has also been achieved on the improvement of regeneration
systems, in particular by somatic embryogenesis. Somatic embryos have been induced
from immature zygotic embryos of various cultivars (Leva et al. 1995), from cotyle-
dons of mature zygotic embryos (Pritsa and Voyiatzis 1999) and from seeds using
radicle segments as explants. Somatic embryogenesis from tissues of elite olive
cultivars has been difficult to achieve and has been reported only for two cultivars,
‘Canino’ and ‘Moraiolo’ (Rugini and Caricato 1995). Secondary somatic embryos
originate from the epidermis or from the first sub-epidermal layer of embryos, mainly
from their basal part. A limited number of cells of the primary explant seems to be
involved in the formation of embryo’s primordia (Benelli et al. 2001).
13.8.2 Genetic Manipulation
Olive explants have been transformed by the use of Agrobacterium rhizogenes

through microprojectile-mediated DNA delivery on sporophytic explants or
zygotic embryos (Mencuccini and Rugini 1993). Even if transgenic callus has
been produced from leaf petioles of in vitro growing shoots, somatic embryos
represent the most suitable tissue for transformation, because they may con-
tinuously develop secondary embryos (Rugini et al. 2000).
Genes used for transformation of olive were mainly rol genes of A. rhizogenes T-
DNA cloned in A. tumefaciens LBA4404. Transformation with the entire T-DNA
of A. rhizogenes may increase rooting efficiency, but only chimeric plants have been
obtained (Rugini 1992). The rolABC genes allow morphological plant character-
istics to be modified, but somatic embryos and plants have been obtained only
from cv. Canino (Rugini et al. 1999), whereas in all other cases no regeneration has
been obtained. Transformation with the osmotin gene increases defence against
fungal pathogens, and olive transgenes have been regenerated and evaluated in
field trials (Rugini et al. 1999). Olive plants expressing the osmotin gene under the
35S promoter have shown reduced growth. Experiments are in progress to trans-
form 3 genes from tobacco (osmotin + chitinase + PR1) in one construct.
Neomycin phosphotransferase II (npt II), which encodes resistance to kanamy-
cin, has been most widely used as a selective marker. To increase public acceptance
of transgenic olives, the procedures of selection should be improved, possibly by
replacing traditional selection markers. The development of methods avoiding
the use of antibiotic-dependent selection or allowing elimination of marker genes
from transformed plants is a research priority in coming years.
Other methods of genetic modification include induced mutations. By irra-
diating rooted cuttings of cv. Ascolana Tenera with gamma rays, dwarf plants
have been obtained (Roselli and Donini 1982). Irradiation of ‘Frantoio’ and
‘Leccino’ plantlets has produced mixoploid mutants showing dwarf habit, self-
sterility and late blooming (Rugini et al. 1996).
13.8.3 Other In Vitro Technologies

Haploid recovery. Unlike the great interest in obtaining homozygous plants to
isolate mutants, identify recessive alleles and facilitating whole genome sequen-
cing, little work has been devoted to generating haploid plants (to be used for
self-fertilization) from in vitro cultured anthers, and no regeneration has been
obtained (Perri et al. 1994).
Polyploids. Tetraploid plants have been produced by irradiation of ‘Leccino’
and ‘Frantoio’ plantlets. Triploid plants have been obtained by pollinating
mixoploid or tetraploid plants with regular haploid pollen.
Protoplast culture. Viable protoplasts have been isolated from hypocotyls,
cotyledons, and leaves of micropropagated shoots, but it has been impossible to
obtain regeneration (Canas et al. 1987).
Somaclonal variation. Somaclonal variation has been observed in mature
olive plants regenerated through somatic embryogenesis of the ‘Frangivento’
cultivar obtained from embryogenic tissue induced on immature cotyledon
portions. Plants have shown two types of variation: BOS (bushy olive soma-
clone), characterized by reduced plant height, leaf, inflorescence and fruit size,
and COS (columnar olive somaclone), characterized by increased plant height,
canopy volume and fruit size. The causes of this variation remain unknown
(Leva and Petruccelli 2007).
13 Olive 415
Cryopreservation. With respect to medium- and long-term conservation, pro-

mising results have been obtained by slow growth storage and cryopreservation. In
‘Arbequina’ 30% survival of shoot tips has been obtained following their desicca-
tion to 30% moisture content and direct immersion into liquid nitrogen (Martinez
et al. 1999). Utilizing vitrification and one-step freezing in liquid nitrogen for shoot
tips excised from in vitro ‘Frantoio’ shoot cultures, Lambardi et al. (2000) have
achieved 15% survival, following re-warming to 408C and plating on re-growth
medium. A higher percentage (38%) of cryopreserved embryogenic cultures was
obtained when using embryogenic cultures such as proembryonal masses and
somatic embryos, and recovered embryogenic tissues showed enhanced prolifera-
tive and morphogenic activity. The encapsulation-dehydration procedure proved
ineffective for cryopreservation (Martinez et al. 1999) but has potential applica-
tions in olive propagation (Micheli et al. 1998). Generally, cryogenic methods can
be applied for long-term conservation of olive germplasm and the establishment of
in vitro repositories, which could safeguard olive biodiversity.
Studies on olive genetic resources have been intensively carried out in recent
years, while a serious gap is envisaged for what concerns breeding activities and
genomic research. Efforts are currently put on by many research groups to
rapidly cover these areas.
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Chapter 14
Safflower
Hans-Henning Mündel and Jerald W. Bergman
14.1 Introduction
Significant contributions to understanding and manipulating domesticated saf-

flower, Carthamus tinctorius L., for improvement of the crop, have been made
by scientists in numerous countries. A sampling of such contributions will be
presented in this chapter. However, two countries stand out in terms of the
amount of research carried out on safflower, namely India and the USA. India
produces more safflower and has more safflower researchers than any other
country. The USA has contributed considerably to the academic training
involving safflower over the past half century, to scientists from around the
world. Contributions from India and the USA will be highlighted in this
chapter.
Cultivated safflower may have its origins from the two related species, wild
safflower, Carthamus oxyacanthus M.Bieb. from Afghanistan and adjoining
countries (e.g. Pakistan) and from the saffron thistle, Carthamus lanatus from
Ethiopia (Chavan 1961).
Using wide-ranging sources, Weiss (1971) determined that safflower,
Carthamus tinctorius L., has been recorded as being grown for centuries in a
wide area covering southern and western China, much of India and westward
across present-day Pakistan, Afghanistan, Iran, Iraq, northern Saudi Arabia,
Kazakhstan, Turkey and numerous other ‘Middle Eastern’ countries; as well
down the Nile Valley of Egypt and Sudan, and Ethiopia. Egyptian mummies
were anointed with a ceremonial ointment using a safflower dye, prior to
binding, 4,000 years ago. Safflower seed packets and garlands of florets were
found with such mummies (Weiss 1971). While the main use of safflower was
H.-H. Mündel (*)

PO Box 21031, West View Mall, Lethbridge, AB T1K 6X4, Canada
e-mail: hhmuend@shockware.com

424 H.-H. Mündel and J.W. Bergman
for foods, for spice and colouring and dying of clothing, it has been known as an
edible oil crop in the ancient Mesopotamian region over 2,000 years ago
(Weiss 1971). In China safflower, introduced around the 2nd century BC
(Chavan 1961), has been used almost exclusively for medicinal purposes by
use of its florets applied topically or in a tonic tea in both ancient and modern
medicine (Li and Mündel 1996). The use of carthamin tablets for food colour-
ing, rouge and medicine have been described in Hebrew writings from the 2nd
century AD, while both European and Japanese pharmacopoeias of the past
listed safflower among their medicines (Chavan 1961; Weiss 1983). Just prior to
the 10th century, the Persian physician-pharmacist, Mesua (originally called
Yahya ibn Masawayh), provided drawings of safflower as recognizable as being
C. tinctorius, for around Baghdad as well as from India (Chavan 1961).
Before the discovery of aniline dyes, benzene derivatives, in the early 19th
century, natural products such as indigo (for blue) and safflower – for yellow,
orange and red pigments from the flower petals – were important sources of
vegetable dyes for colouring fabrics (Knowles 1982).
Since the 20th, century and probably as early as the 19th century, safflower
has been grown mostly for its edible oil in India, where it is cultivated in many
states (Chavan 1961). Commercial oil production from safflower began in the
United States of America (USA) in Nebraska in the 1940s and expanded to
California by 1950 where production dominates as well as in the Northern
Great Plains of eastern Montana and North Dakota (Knowles 1980). Sizable
production also occurs in Mexico and in much smaller areas in numerous other
countries (Li and Mündel 1996). Almost half of the worldwide production of
safflower, or close to 1.5 million ha is grown in India, in many states mostly in
central and southern regions; the remainder is grown typically in drier regions
of many countries (Sastry 1997).
14.3 Species Groupings Related to Breeding of Cultivated

Safflower
A botanical classification for safflower, in both Latin and English, is given by
the USDA (Anon. 2007a) as follows: Kingdom Plantae – Plants; Subkingdom
Tracheobionta – Vascular plants; Superdivision – Spermatophyta – Seed
plants; Division Magnoliophyta – Flowering plants; Class Magnoliopsida –
Dicotyledons; Subclass Asteridae; Order Asterales; Family Asteraceae – Aster
family; Genus Carthamus L. – distaff thistle.
As the topic of this chapter is safflower breeding, only the species of most
likely relevance to the actual breeding will be discussed in detail. Khidir (1969)
described the probably most basic ploidy condition of Carthamus as the one of
2n = 24 chromosomes, which is predominantly outcrossing with occasional
selfing. Beside the cultivated C. tinctorius, with 2n = 24, species that produce
fertile natural as well as artificial F1 and F2 hybrids with the cultivated species
14 Safflower 425
are: C. oxyacanthus M.Bieb., known as ‘wild safflower’, C. palaestinus Eig and

C. persicus Desf. ex Willd. (Syn. = C. flavescens Spreng) (Ashri 1974). Thus,
only these three wild species of Carthamus, which are closely related to the
cultivated safflower, contribute variability to the gene pool from beyond the
species C. tinctorius itself. While cultivated safflower, C. tinctorius, is self-
compatible and self-pollination as well as out-crossing occur naturally,
C. oxyacanthus is a mixture of self-incompatible and self-compatible types;
C. palaestinus is a self-compatible species; and C. persicus is entirely self-
incompatible (Knowles 1982).
Ashri and Knowles (1960) classified the genus Carthamus into four sections,
based on chromosome numbers. More recently however, López-González
(1989) developed a classification system, based on anatomical, chorological
(biogeographic, related to distribution) and biosystematic information. In this
system, the two genera Carthamus and Carduncellus are replaced by four new
genera: Phonus, Lamottea, Carthamus and Carduncellus. Species of the three
genera Phonus, Lamottea and Carduncellus are classified as perennial and have
24 chromosomes in their genomes, whereas the newly circumscribed genus
Carthamus contains only annual species, and has members of 20, 22, 24, 44
and 64 chromosomes, including several putative allopolyploid species. The
geographical distribution for Carthamus is west and central Asia as well as in
the Mediterranean region; of the four genera, only this new genus Carthamus is
further subdivided into sections, with the species indicated. Section Carthamus
has 24 chromosomes and includes the following species: C. curdicus Hanelt,
C. gypsicola Ilj., C. oxyacanthus Bieb., C. palaestinus Eig, C. persicus Willd. and
C. tinctorius L. Section Odonthagnathius (DC.) Hanelt (incl. Sect. Lepidopappus
Hanelt) has 20 or 22 chromosomes and includes the following species:
C. boissieri Halácsy, C. dentatus Vahl, C. divaricatus Beguinot & Vacc.
(with 22 chromosomes), C. glaucus Bieb., C. leucocaulos Sm. and C. tenuis
(Boiss. & Bl.) Bornm. Section Atractylis Reichenb., with a presumed x number
of 11, contains numerous polyploids, including the following species: C. lanatus
L., C. creticus (C. baeticus [Boiss. & Reuter] Nyman) and C. turkestanicus M.
Popov. Subsequent molecular studies have clarified the problem of the generic
limits of Carthamus, confirming Carthamus as a natural group, using molecular
phylogenies based on DNA sequences (Vilatersana et al. 2005). Analyses of
genome size showed that the species of section Carthamus form a distinct
cluster, with the most invasive Carthamus spp. exhibiting an increased genome
size but a decreased chromosome number, with respect to the other taxa of the
genus (Garnatje et al. 2006).
While numerous studies on crossing among Carthamus species and
associated cytogenetics have been carried out in the past, relatively few actual
crosses have been recorded towards improvement of the domestic safflower
using the wild or weedy relatives. A sample of these is described below. The
three wild species mentioned above, also having 12 pairs of chromosomes, all in
the new genus Carthamus subsection Carthamus, namely C. oxyacanthus,
C. creticus and C. palaestinus, have basic fatty acid compositions similar to
those for the commercial C. tinctorius safflower types. However, some

collections could be used to raise levels of palmitic acid (Knowles 1972).
While the lack of seed dormancy can be a shortcoming in commercial safflower,
where continuous rainfall after maturity result in seed germinating in the head,
crosses with C. palaestinus have been successful in transferring dormancy to the
domesticated species (Kotecha and Zimmerman 1978). Crosses of common
safflower to C. persicus indicated that cold tolerance in the early growing
stage could be transferred to the domesticated species (Zimmerman and
Buck 1977).
Over the decades, various researchers conducted safflower collecting

expeditions, with perhaps the most comprehensive being those of Paulden
F. Knowles in 1957 (Knowles 1959) and 1964–1965 (Knowles 1965). These
expeditions resulted in the deposition of major accession items in the USDA
World Collection of Safflower, which has become a major source for safflower
breeders and germplasm researchers around the world. Access to this material is
available via the Regional Plant Introduction Station in Pullman, Washington,
USA. In 1958, Knowles collected samples from 14 countries from India
westward through the Middle East, North Africa, and southern Europe and
in 1964–1965, from nine countries from India in the east, westward to include
Egypt, Sudan and Spain, which includes both cultivated, wild and weedy
safflower species.
Some of the major national safflower collections, evaluations and documen-
tations, include those of China, India and the USA.
The Safflower Research Group of the Beijing Botanical Garden of the
Chinese Academy of Sciences, with the support of IBPGR since 1989, recorded
a total of 2,051 accessions from 49 countries, and 465 specimens from within
China. Extensive evaluations, based on complete grow-outs of the germplasm
at Beijing, have been reported in English (Li et al. 1993). Similar evaluations at
Urumqi, in Xinjiang in western China, have been reported in Chinese
(Wang Zhaomu and Fan Lin 1991; Wang Zhaomu 1993).
Safflower research in India is coordinated from Solapur, in Maharashtra
State, where the Germplasm Management Unit (GMU) is the major repository
for world safflower germplasm in India, with 6,115 accessions assembled from
38 countries (Mehtre et al. 1995). The GMU in cooperation with the Project
Coordination Unit (Safflower) at the Mahatma Phule Agricultural University
and the Directorate of Oilseeds Research (DOR) in Hyderabad, has
coordinated the systematic safflower germplasm handling and documentation
since the early 1980s when characterization of indigenous and exotic collections
and elimination of duplicate entries reduced the original 9,000 accessions to
1,196 (Rao et al. 1991).
14 Safflower 427
Zhang and Johnson (1999) compiled a directory of safflower germplasm

collections, listing 15 countries as having Carthamus collections. Only those 11
countries with more than 10 accessions and permitting exchange of germplasm,
even if somehow restricted, are included in Table 14.1. Where additional infor-
mation could be obtained (either from researchers in those countries or the
on-line IPGRI Directory of Germplasm Collections [Anon. 2007b]), such
numbers, with a date, are included. Numbers of accessions refer to C. tinctorius
unless otherwise stated. Only such species that are expected to hybridize with
C. tinctorius and produce fertile offspring are included here.
As summarized by Li and Mündel (1996), safflower seed is an ‘orthodox’ seed
in terms of its storage behaviour, viability of seed is maintained best by storing
well-dried seed at low humidity and at low temperatures. In dry environments,
safflower seed equilibrates at around 6–7% moisture. Based on the International
Board for Plant Genetic Resources (IBPGR) guidelines established, ‘medium-
term storage’ can be accomplished by storage at 48C and 30% relative humidity;
‘long-term’ storage can be effected at –208C. To the extent possible, with the
financial resources provided, centres storing the collections outlined below use
those or similar sets of conditions. Unfortunately, however, a number of collec-
tions are stored at ambient temperature and humidity. This results in a great
potential loss of viability and accumulation of mutations as viability is reduced.
It should be noted that C. oxyacanthus, which has been introduced to North
America, is now considered a noxious weed in the USA, appearing on the USDA
Federal Noxious Weed List of banned plants on June 7, 1999 (Anon. 1999).
Indeed it is known as a serious weed in its native habitat of northern India,
Pakistan, west to Iraq (Ashri and Knowles 1960) and in Australia where it had
also been introduced (Weiss 1983). Thus, safflower breeders are cautioned.
In the USA, Johnson et al. (1999) studied one thousand safflower accessions
to: (1) provide oil and meal evaluation information for a major portion of the
United States Department of Agriculture (USDA) safflower (Carthamus
tinctorius L.) collection, (2) compare ranges, variances and means between 203
core and 797 non-core accessions, and (3) determine if region of origin could be
differentiated based on accessions’ oil and meal characteristics. While the core
was not fully representative of the non-core accessions, it captured a large
fraction of the diversity in oil and meal factors present.
In India, Dwivedi et al. (2005) developed a core subset of safflower based on
12 morphological descriptors and geographic information on 5,522 safflower
accessions; stratifying the accessions by country of origin, and data on
12 descriptors. About 10% of the accessions were randomly selected from
each of the 25 clusters to constitute a core subset of 570 accessions. Mean
comparisons of the descriptors indicated that the genetic variation available
for these traits in the entire collection was preserved in the core subset. This core
subset thus selected provides an opportunity to evaluate agronomic and seed
quality traits and resistance to abiotic and biotic stresses and to identify diverse
germplasm with beneficial traits for enhancing the genetic potential of
safflower.
428
Table 14.1 Safflower germplasm collections of countries permitting exchange

Accessions
Country (sources) (date) Institute Storage conditions Availability
Australia (from 27 countries) 486 (2003) Australian Temperate Field Crops Collection Horsham, Medium- & long-term Small amounts
Victoria
Canada (duplicates in India) 34 Plant Gene Resources of Canada, AAFC, Saskatoon Long-term Yes
China (from over 50 countries) 2784 Inst. of Oil Crops Res. YAAS, Kunming, Yunan- duplicates Short-term Partial
of most at CAS, Beijing and XAAS, Urumuqi, Xinjiang
178 Inst. of Crop Germplasm Resources, CAS, Beijing Long-term Mutual exchange
Ethiopia (indigenous) 197 (2007) Inst. of Biodiversity Cons. and Research, Addis Ababa Short-term MTA needed
Germany 167 (2007) IPK, Genebank, Gatersleben Long-term Yes
C. lanatus 31 (2007) IPK, Genebank, Gatersleben Long-term Yes
118 (1995) Fed.Centre for Breeding Res. of Cultiv. Plants (BAZ), Not known Not known
Quedlinburg
India 2393 Nat. Bureau of Plant Genetic Res., New Delhi (Also: Long-term Yes
Regional Station of NBPGR, Akola)
Mexico (from 22 countries) 1504 Instituto Nacional de Investig. Agricolas, Iguala Medium- term Restricted
120 Inst.Nac. de Investig. Forestales, Agricolas y Pecuarias, Not known Not known
Mexico City
Romania 31 (2002) Gene Bank of Suceava, Suceava Medium-term Restricted
Russia (from over 40 418 (2002) NI Vavilov Inst. of Plant Industry, St. Petersburg-duplicates Medium-term Yes
countries) at Kuban Expt. Station of VIR, Krasnodar Region and
Uzbek Res. Inst.of Plant Ind., Tashkent, Uzbekistan
Turkey (indigenous) 27 Aegean Agricultural Research Inst., Menemen, Izmir Medium- & long-term Yes, following gov’t
regulations
United States of America 2288 Western Regional Plant Introd. Station, USDA-ARS, Medium- term Yes
Carthamus lanatus 7 Pullman, Washington - duplicates of most held at Fort
Carthamus lanatus ssp. Collins, Colorado
turkestanicus 4
Carthamus oxyacanthus 90
Carthamus palaestinus 3
H.-H. Mündel and J.W. Bergman
14 Safflower 429
Genetic improvement of safflower, though a self-pollinating species but

showing variably low to considerable outcrossing, has relied to a great extent
on exploiting the existing variability in cultivated varieties and land races, and
to a limited extent also on crosses with closely related species (Ashri and
Knowles 1960).
In India, where more safflower is grown than anywhere else in the world,
safflower landraces have been cultivated over millennia. Thus it is not
surprising that there are and have been more safflower breeders and other
researchers in India than in any other country, trying inter alia to improve the
productivity of local safflower. Screening for resistance to a number of the
common safflower diseases has generally not directly resulted in resistant
varieties in India, but has provided useful resistant or moderately resistant
germplasm to use (Mündel and Huang 2003). The All India Coordinated
Research Project on Oilseeds (AICORPO) was initiated in the late 1960s and
from the early 1970s on included also safflower. This project included mainly
public research but also a private research center, the Nimbkar Agricultural
Research Institute (NARI), in Maharashtra State, in addition, the Maharashtra
Hybrid Seed Company (MAHYCO) includes safflower as one of its crops.
Aside from developing high yielding cultivars for the diverse regions growing
safflower, these programs have developed a wide range of plant types, such as
appressed, semi-compact, spreading, in both spiny and non-spiny versions, with
a result of 18 high-yielding varieties and four hybrids of regional and
multi-rgional importance (Sujatha 2006). The first-ever spineless variety,
JSI-7, was developed in Indore and released in 1990, to facilitate the introduc-
tion of safflower in non-traditional areas of safflower cultivation, realizing that
harvesting is done manually (Anjani et al. 2006). The same report mentions the
release of the spineless NARI-6 in 2000 by NARI, providing dual income to
farmers, as the florets can easily be collected from non-spiny safflower after the
crop matures and sold for food and textile dye.
In the USA, safflower breeding has been carried out in numerous states,
from the 1940s on in Utah, Nebraska, Arizona, then California with public
breeding at the University of California at Davis, and several private companies
being involved. From the 1960s on, a major safflower breeding program was
commenced in eastern Montana, at Sidney. A series of varieties was released
with resistance to alternaria leaf blight (caused by the fungus Alternaria
carthami Chowdhury) (Bergman et al. 1985, 1987, 1989). These varieties were
developed by mass-selection of resistant plants from crossing of existing
varieties in a disease nursery initiated in the early 1960s. In anticipation of
the need for high oleic safflower varieties, this program developed a series
of such varieties named Montola and year of release, starting with Montola-
2000 (Anon. 2007c). As infection with safflower rust (caused by Puccinia
carthami Cda) at the seedling stage can cause serious yield reduction in
warm soils, five improved safflower lines (PCA, PCM-1, PCM-2, PCN, and
PCOy) have been developed, each carrying a different dominant gene for rust
resistance (Zimmer and Urie 1970). Also stem and root rot resistant varieties,
resistant to Phytophthora drechsleri Tucker, have been developed over the
decades, including Gila, US10, VFR-1, Dart, and most California cultivars
(Mündel and Huang 2003). Anecdotal evidence from numerous countries
indicates that the highly-variable variety Gila (Dave Rubis – personal commu-
nication), released from the Arizona program in 1958, has in following decades
been grown in more countries and covering more area than any other single
variety. From the 1970s on, some of the varieties of the private breeding
company, SeedTech, headquartered in Woodland, CA, have dominated com-
mercial production areas in the US. This included specially S-208 and S-541,
with their high-oleic variety, S-317 being produced mainly in a rather confined
area in California.
Contributions to disease resistant varieties and germplasm have also been
made by safflower programs in countries with very modest safflower breeding
programs. For example, in Australia the cultivar Sironaria was developed in a
backcrossing program to Gila, selecting plants to incorporate resistance to
races of A. carthami (Harrigan 1987); and in Canada, Saffire was developed
by mass-selection from a bulk derived from selections from India, having good
field resistance to head rot, caused by Sclerotinia sclerotiorum (Lib.) de Bary
(Mündel et al. 1985).
Important examples of major screening of safflower germplasm against
insect and diseases, as well as describing in detail thousands of lines are those
from China and Israel, in which important germplasm lines have been identified
for use in breeding programs world-wide (Ashri 1971; Li et al. 1993; Wang
Zhaomu 1993).
Genetic improvement in safflower has been commonly aimed at improvement

of yield, oil and other agronomic characteristics, including resistance to dis-
eases, insects and abiotic stresses.
Over the decades, one of the common breeding goals has been and continues
to be the increase of oil content. One way this has been attempted is by the
reduction in the ratio of seed hull of the achenes to the total seed. While a typical
commercial safflower seed may be white (Fig. 14.1) as shown in Saffire, various
genetic variations have been developed with resultant reductions in the see coat,
and hence increases in oil content, ranging from 42 to 50% in California
(Knowles 1982). Thus, partial hull (Fig. 14.2), reduced hull (Urie and Zimmer
1970) (Fig. 14.3), and striped hull (Fig. 14.4) can be used for commercial
varieties.
14 Safflower 431
Fig. 14.1 White seeds of

Saffire safflower
Fig. 14.2 Partial hull seeds
Typically the fatty acid composition of safflower has been over 70%
linoleic acid, a polyunsaturated fatty acid, and with also a significant
percentage oleic acid, a monounsaturated fatty acid. The genetics of the
fatty acid composition has been determined and manipulated for different
uses. Over the past decade high oleic acid safflower is favoured by nutri-
tionists and for frying, hence by the commercial trade. Knowles (1972)
summarized information on the genetics of fatty acid composition. Three
alleles at one locus govern the levels of linoleic and oleic acid, with the
intermediate levels being temperature sensitive: linoleic contents being high
Fig. 14.3 Reduced hull seeds
Fig. 14.4 Striped hull seeds
under cool temperatures and oleic contents being high under high tempera-
tures. As the genetic control of fatty acid composition in safflower is
expressed in the embryo (Knowles 1983), F2 segregations can be determined
from single seeds on an F1 plant.
The All India Coordinated Research Project on Safflower, has identified the
following goals as their thrust for the Xth Plan (2002–2007) of the Indian
government (Damodaram 2006):
14 Safflower 433
– Development of spineless varieties with high yield and high oil content
possessing resistance to Alternaria wilt and aphids.
– Exploration of heterosis through the development of CMS, maintainer
and restorer lines.
– Development of seed production technology for the hybrids already
released through GMS background.
– Wilt and Alternaria disease management and aphid management through
IPM technologies.
– Development of region-specific crop production technology for higher
production.
14.7 Crossing Techniques and Breeding Methods

14.7.1 Crossing Techniques
Safflower is a predominantly self-pollinating crop. However, depending on

the genetics and environmental conditions, outcrossing may be considerable
(Li and Mündel 1996). A diversity of bees, as well as other insects, are
attracted to safflower pollen and nectar, and thus contribute to outcrossing.
Thus, to ensure planned crossing, flowers must be emasculated prior to
pollen shedding.
While specific procedures in use by different breeders may differ somewhat,
the following provides a generally acceptable procedure for crossing safflower.
In this case a greenhouse or phytotron is assumed for crossing, thus insect
contamination of the pollen parent is obviated. As indoors heads may be fewer
than in the field, staggered planting over several weeks is advisable, to ensure
pollen availability when stigmas of the designated females are receptive.
Considerable variation may be observed in different safflower germplasm
lines or varieties. The calyx is removed and at least the first (outermost) whorl
of flowers, which is typically female-sterile (Knowles 1980) when the first
individual flowers have elongated and are about to or have just appeared
above the bracts (Fig. 14.5). Once the first flowers have elongated, which can
occur inside the calyx in some material, typically anthers have dehisced and
pollination has already been effected.
In preparation for crossing, the outer corolla tubes of each flower are gently
broken by holding with slender tweezers and twisting slightly until a snap is felt
just below the origin of the anther filaments. Then the upper part of the corolla
tubes with the five attached fused anthers is slid upwards and outwards over the
style and stigma, ensuring that the latter is not damaged nor breaking off the
style. This can best be accomplished by taking the tip of the closed corolla lobe
with the tweezers and gently pulling it up and off the rest of the flower head
(Fig. 14.6). Immature buds in the centre of the head should be cut off with the
tweezers.
Fig. 14.5 Developmental stage for preparing flowers for crossing
Fig. 14.6 Emasculation by

sliding corolla tube upwards
The next day, if the styles have elongated considerably (Fig. 14.7), indicating
that the stigma would be receptive, pollen can be added by using whole flowers
from the pollen parent (Fig. 14.8), if adequate flowers are available. Pollination
can commence an hour or two after daylight, natural or artificial, and continue
14 Safflower 435
Fig. 14.7 Elongated styles
Fig. 14.8 Pollination

for several hours. If pollen is in short supply, one pollen-rich style of the
pollen-parent may be used to pollinate a number of stigmas of the female
emasculated parent. The pollinated capitula is covered with plastic bags,
appropriately labeling the male and female with dates of emasculation and
pollination on small tags. After a few days the successful crosses will set seed
and to prevent high humidity build-up inside the head, at least the top of the
plastic bag can be cut off, or the bag can be removed completely (Fig. 14.9). As
soon as seeds mature they may be harvested. Delays may result in crossed seeds
falling off.
Fig. 14.9 Head of crossed

seeds
At the NARI in Phaltan, India, a mass-emasculation technique was devel-

oped (Deshmuk and Ranga Rao 1991). At flower initiation, 5–10 fully devel-
oped capitula from the top four or five branches of each plant are covered with
polythene bags in the field. The remaining branches are pruned off. Tempera-
ture and moisture build-up inside the bags prevents dehiscence of anthers.
When 50% flowering is attained, the bags are removed, flowers are pollinated
with the desired pollen source and the bags are again closed. This may be
repeated for three successive days. Once flowering is complete, tissue paper
bags are used to replace the polythene bags to reduce moisture and hence
disease accumulation in the head. This technique has been shown to permit
up to 10 times as many crossed seeds to be produced in the same time as
14 Safflower 437
emasculation of individual florets. However, this mass-emasculation technique

is only effective at the moderate temperatures of December and early January at
Phaltan. At higher temperatures, the pollen is sterilized in the bags.

14.7.2.1 Pure-Line Selection and Mass Selection
Pure-line selection is the oldest and most extensively practised method of crop
improvement for safflower. Weiss (1983) mentions that the term ‘pure lines’ was
then used rather loosely and that many of the varieties resulted from selections
in early generations following a cross. Thus, such varieties will in fact be
mixtures of similar types, appearing quite uniform under commercial produc-
tion. In India, 16 out of the 28 varieties released for commercial production
since N-630 was released in 1942, have been developed by carrying out selection
in the local landraces (6) and selections in varieties or germplasm lines (10 for
these two groups together) (Nimbkar, personal communication 2007).
Mass selection from fields naturally infested with a multitude of diseases has
been used in Montana, USA, to develop cultivars with improved field resistance
to several diseases, including leaf blight caused by Alternaria carthami and
bacterial blight caused by Pseudomonas syringae van Hall. This was followed
by crossing to commercial varieties and resulted in such varieties as Oker,
Hartman and Girard (Bergman et al. 1985, 1987, 1989).
14.7.2.2 Pedigree Breeding

Plant breeders of safflower have generally used variations on the pedigree
method for handling segregating generations (Knowles 1989), selecting for
highly heritable characters (e.g. early maturity, disease resistance) beginning
from single F2 plants. To improve seed yield, oil content and other desirable
traits in safflower, breeders have often followed the pedigree method. Starting
with the release of A-1 in Annegiri in 1969, almost a dozen safflower varieties
have been released by both government and private breeders in India using the
pedigree method (Nimbkar, personal communication 2007).
14.7.2.3 Backcross Breeding

Backcrossing has been used to introduce specific characters, especially disease
resistance, into otherwise good commercial cultivars. For example for transfer-
ring the Fusarium oxysporum (wilt) resistance genes to the wilt-susceptible
variety Nira (Singh et al. 2003) in India. The wilt resistant varieties developed
by using the backcross method are presently in multilocation evaluation for
assessing their adaptability and yielding ability and to identify the most suitable
one for commercial production.
14.7.2.4 Recurrent Selection

Recurrent selection programmes have also sometimes been used in safflower
improvement, when the proper tools were available. For example, in a
programme begun in 1970 in Arizona, USA, Rubis (1981) used structural
male sterility associated with the thin-hulled gene (th th) to enforce outcrossing
and produce lines highly resistant to root rot caused by Phytophthora spp. The
thin-hull gene has shown crossability of 98–100%. Flooding, along with high
temperatures at flowering time, following moisture stress imposes a very strong
selection pressure for resistance to phytophthora root rot. Enforced
fertilization by pollen among the surviving plants resulted by the use of this
thin-hulled gene. Thus, a complete cycle of recurrent selection was achieved in a
single year. The survival rate of safflower plants increased from 14% in the best
plots in 1972 to 85% in 1980, while the survival rate of the check variety (Royal)
was 0%.
Based on a genetic analysis of seed yield, oil content and their components of
four Indian and seven US safflower lines in an 11 11 diallel cross, Ramachan-
dran and Goud (1981) suggested a combination of breeding methods, such as
biparental mating followed by reciprocal recurrent selection, for the simulta-
neous improvement of seed yield and oil content.
14.7.2.5 Hybrid Breeding

As safflower is largely self-pollinated, though outcrossing does occur naturally,
significant heterosis is expected for such economically important factors as
yield and oil content. Thus, efforts to produce commercially viable hybrids
has been ongoing for many years.
Genic male sterility, identified in safflower by Heaton and Knowles (1982),
has been considered for use in hybridization to produce high-yielding cultivars.
However, manual removal of male-fertile plants in crossing blocks has gener-
ally made this procedure prohibitively expensive where labour costs are high.
In studying the effectiveness of early-generation selection for yield by using
various yield components in hybrid safflower, Patil et al. (1994) used only fertile
plants of 25 hybrids developed with parents selected on the basis of good
general combining ability. The segregating progenies studied included five
dominant genetic male sterile parents crossed with 10 male parents. While test
weight and seeds per capitulum were ineffective in selecting for yield, capitula
per plant and individual plant yield were useful early-generation selection
criteria.
USA
Rubis (1967) identified a form of structural sterility, linked with the thin-hull
mutant. However efforts to develop hybrids using this system failed because of
a high percentage of selfed female plants, which adversely affected yield
14 Safflower 439
(Sujatha 2006). Furthermore, seeds with the thin-hulled character are readily
damaged on commercial harvesting.
Hill (1991), working for Cargill at the time, initiated a program to produce
commercially viable safflower hybrids in 1972. This system for the development
of cytoplasmic male sterile (CMS) lines relied on the use of the wild safflower,
C. oxyacanthus as female and the domestic safflower, C. tinctorius as restorers
of CMS and as recurrent males. Because of dominance of the restorers (R-lines),
F2 populations were required from each of the backcross females starting at
BC3 and selecting only the sterile plants. Hybrid testing began in 1983, with
gradual improvements in oil content and absence of pappus on the seeds.
Various partnerships had been developed between A.B. Hill and his Safftech
Hybrid Safflower company and initially two other California safflower
companies developing safflower varieties, SeedTec and Cal West Seeds
(Hill 2005), then mainly with Montana-based Safflower Technologies Interna-
tional. Yield increases in the hybrids had been recorded from multi-location
trials and birdseed safflower hybrid commercialized. High-oil, with 44% oil,
B-lines are expected in test hybrids by 2008 or 2009. This system is not available
to the public.
India
With significant heterosis of up to 177% for seed yield and 80% for oil being
reported for safflower, led to the development of both dominant and recessive
genetic as well as cytoplasmic male sterility systems in India (Sujatha 2006;
Singh and Nimbkar 2007). Five hybrids, including dominant GMS, recessive
GMS and CMS systems, have been released in India over the past 10 years
(Nimbkar, personal communication 2007). Aside from improvements in yield,
these hybrids variously have moderate to high field resistances to Alternaria wilt
and leaf spot and three show tolerance to the black aphids as well.
Hybrid seed production, using the genetic male sterility systems, necessitates
labour-intensive roguing before flowering of 50% male fertile (MF) plants
appearing in the female parent (GMS line) (Sujatha 2006). The roguing of
MF plants in the hybrids based on non-spiny genetic male sterile lines is
economically viable. However, in case of spiny genetic male sterile lines due
to presence of spines on the plants, roguing of MF plants is cumbersome and
tedious and hence is commercially not feasible.
The development of cytoplasmic-genetic male sterility system is underway at
the NARI, Phaltan, Maharashtra (Singh et al. 2001) and at the Directorate of
Oilseeds Research (DOR), Rajendranagar, Hyderabad (Anjani 2005). The
system developed in Hyderabad employed an interspecific cross between
C. tinctorius and C. oxyacanthus, with the latter being the donor of the sterile
cytoplasm. Single gene segregation for male sterility to male fertility, showed
dominance and that C. tinctorius possesses a nuclear fertility restorer gene (Rf),
with hybrid progeny carrying the cytoplasmic male-sterile (CMS) cytoplasm of
C. oxyacanthus. Thus male sterility occurred with the homozygous recessive
condition (rfrf) in a sterile C. oxyacanthus cytoplasm background, but not in the

normal cytoplasm of C. tinctorius. The utilization of a CMS system for hybrid
development at these institutes is hindered due to the unavailability of suitable
male sterility maintainer genotypes. However, another Maharasthra private
seed company, MAHYCO, has not only developed the male sterility maintainer
genotype for the sterile cytoplasm but has developed and released the first
CMS-based safflower hybrid MRSA-521, expressing high wilt resistance, for
commercial cultivation in India during 2006–2007. With the development of
CMS-based hybrid MRSA-521, released by MAHYCO in 2006, it is expected
that hybrid seed will be made available much more cheaply to the producers as
compared to the seed of GMS-based hybrids in safflower. This should lead to
the rapid expansion of hybrid safflower area in India. In India, the area under
hybrid safflower is increasing gradually, however it still comprises less than 5%
of the total safflower grown in the country.
Apart from the development of GMS and CMS systems, the NARI has
developed thermosensitive genetic male sterility (TGMS) in safflower
(Nimbkar, personal communication 2007). In this system, during winter,
which is the normal safflower-growing season, TGMS lines show complete
male sterility, with temperatures ranging from 10 to 328C, behave as fertile
genotypes when grown under summer conditions, with day temperatures
during pre flowering and flowering ranging from 19 to 428C. The hybrids
developed from such lines show complete fertility in both winter and summer
seasons. The TGMS hybrids have exhibited an average increase if yield of
15–20% over the best GMS hybrids. Multilocation evaluation of
TGMS-based hybrids will be started in the winter 2007–2008.
Safflower offers potential in the use of Agrobacterium-mediated gene transfer.

Agrobacterium tumefaciens-mediated transformation and regeneration of
transgenic safflower using the variety Centennial was accomplished by Ying
et al. (1997). However, root regeneration was at a low percentage. A protocol
for regeneration of plantlets with well-developed root systems was developed by
Tejovathi and Anwar in 1993.
The genetic modification of crop plants for use as protein factories has been
pursued for nearly a decade as a potential method of meeting the high volume
demands made on the pharmaceutical industry. Safflower is a host species with
growing appeal for genetic modification for biopharming for the production of
pharmaceuticals, proteins, enzymes, and safflower oil modifications for
specialty oil markets.
A Calgary, Alberta, Canada-based company, SemBioSys Genetics Inc., has
found safflower to be a very attractive host for the production of high value
proteins such as pharmaceuticals and industrial enzymes. SemBioSys
14 Safflower 441
genetically transforms safflower tissue so that the proteins of interest will

accumulate in the seed of the mature transgenic plant. The patented Strato-
someTM Biologics System involves the genetic attachment of commercially
viable target proteins to oleosin, the primary protein coating the oil-containing
vesicles (oil bodies) of the seed (Fig. 14.10). This attachment allows the target
protein to be purified along with the oil body fraction which floats to the surface
of a ground seed/water slurry upon centrifugation (Fig. 14.11) (van Rooijen
Fig. 14.10 Oil bodies

(oleosomes) and protein
bodies in cells of oil-bearing
tissue
Fig. 14.11 Schematic representation of purification of recombinant proteins produced in

genetically engineered oil body membranes
et al. 1992). This initial purification step gives the system a favourable process
advantage over other transgenic plant systems. In addition to the purification
advantage of the StratosomeTM Biologics System, the attachment of proteins to
the oil bodies of safflower has been shown to stabilize intracellular
accumulation of foreign proteins as well as providing a useful attachment
matrix and delivery benefits for downstream applications.
The uncontained, outdoor production of so-called ‘Molecular Farming’
crops such as SemBioSys’ GM safflower offers the potential for economical,
huge-scale production of pharmaceuticals, indeed ‘molecular pharming,’ and
industrials but, understandably, must be regulated for proper material
confinement. The Canadian Food Inspection Agency (CFIA) (in Canada)
and the United States Department of Agriculture (USDA) (in the USA) have
placed a host of restrictions on the numerous crops that are used for Molecular
Farming purposes but many of the features possessed by safflower make it a
lower risk production platform. Several inherent agronomic qualities such as a
low tendency to weediness, low seed dormancy and the large degree of
self-pollination translate into a system that is much easier to confine so that
target products don’t mingle with food or feed. The role that safflower plays in
North American agriculture also lends benefits to its use as a pharmaceutical
factory. Safflower is not a major food or feed crop in North America, acreages
are relatively low and there are no weedy relatives with which it can cross to
produce fertile hybrids. As the tiny molecular farming industry grows in North
America, safflower has been receiving increasing attention as a host crop with
great appeal.
SemBioSys Genetics, Inc. has formed strategic partnerships with other
companies to utilize genetic modified safflower varieties for products that
include GM varieties that accumulate omega-3 and omega-6 fatty acids,
human insulin, carp growth hormone, and apolipoprotein-A1 for the treatment
of cardiovascular disease. Commercial production of these GM varieties is
expected to be accomplished in the next several years and will stimulate further
advances in safflower genetic engineering and biotechnology. A comprehensive
review article on the ‘Advances in Safflower Biotechnology’ by Sujatha (2007)
covers the topic in more detail for interested readers.
Maintaining genetic purity of safflower varieties starts with breeder seed to

supply a pure seed source for certified classes of seed that meet seed certification
standards. The seed originators supply a statement of the variety’s origin,
breeding procedure used in development, and detailed description of the plant
and seed characteristics to seed certifying agencies. Any variants in the variety
must be stated so field inspectors can recognize the variation from crop
mixtures. The number of generations through which a variety may be multiplied
14 Safflower 443
should be specified by the originator but usually will not be allowed to exceed
three generations beyond breeder seed. The foundation class of seed must be the
progeny of breeder or foundation seed. The registered class of seed must be the
progeny of breeder or foundation seed and may be omitted by the seed
originator. The certified seed must be the progeny of breeder, foundation, or
registered seed.
Safflower may not be considered for certification if planted where safflower
has been grown the previous two crop years. Seed certifying agencies recom-
mend that safflower be planted on land immediately following a separable crop.
Additionally, safflower is not recommended to follow other oilseed crops.
The minimum isolation requirement for pure seed production of safflower is
at least 400 m from any other variety or non-certified field of safflower. The
recommended minimum isolation requirement for pure hybrid seed production
is 4.83 km. Off-type plants or identifiable variety mixtures must be removed
prior to bloom or before pollination occurs. Specific field standards to pass field
certification depend on the certified class of seed. Field standards restrict the
number of plants of other varieties (none to 1 per 1,000 plants), inseparable
other crops (none to 1 per 3,000 plants), noxious weeds (none). Safflower seed
standards for certification specify a seed purity of 98% with a minimum
germination of 80% and limit the percent inert matter (2%), other crop seeds
(0.1–0.10%), weed seeds (0.01–0.10%) noxious weed seeds (0%), sclerotia
(1 seed per 0.45 kg), and seed moisture of 8% or less.
Producers are responsible for cleanliness and maintenance of all equipment
and storage facilities used in the planting, harvesting, and storage of the
safflower. Approved seed conditioners are usually required for cleaning all
certified classes of seed. Seed cleaning facilities must condition seed without
introducing other crop or variety mixtures in cleaning, treating, handling and
bagging each seed lot of safflower.
Pure seed production growers should rely heavily on herbicide, insecticide,
and fungicide control of safflower pests as an integral and necessary part of pure
seed production. The following herbicides are labeled for weed control in
safflower (USA): paraquat, Eptam (EPTC), trifluralin, sonalan, metolachlor,
clethoderm, and sethoxydem. Azoxystrobin is labeled for foliar application to
control Alternaria leaf spot. Insecticide and fungicide seed treatments and foliar
applied treatments are available and recommended to control insects and
diseases when potential problems may occur in pure seed production fields.
The Montana Seed Growers Association (2006), the North Dakota State
Seed Department (1986), and the California Crop Improvement Association
(2003) seed certification standards were used as references for this section.
Relatively minor variations are expected in these and other jurisdictions.
Acknowledgments We wish to sincerely thank the following, for providing information

related to safflower breeding at their respective institutes and/or countries: M. Sujatha
(DOR, Hyderabad, India), Nandini Nimbkar (NARI, Phaltan, India) and R.C. Johnson
(USDA-ARS, Pullman, Washington, USA).
The following are thanked, for providing information on Carthamus genetic resources held
in their respective institutes: A. Diederichsen (PGRC-AAFC, Saskatoon, Canada),
H. Knüpffer (Genebank-IPK, Gatersleben, Germany).
And we also wish to thank Rick Keon (SemBioSys, Calgary, Canada) for providing us
information/updates on safflower biotechnology related to breeding.
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Chapter 15
Poppy
Jenö Bernáth and Éva Németh
15.1 Introduction
Poppy (Papaver somniferum L.) has been utilised and cultivated since prehis-
toric times (Tétényi 1997). The narcotic and nutritive values of its products were
recognised in ancient Egypt, Greece and Rome. Hippocrates (460–377 BC) was
among the first to emphasise the medical advantages of poppy and its prepara-
tions. He also recognised the nutritive property of poppy seeds. Poppy spread
from its Central Asian gene centre through the Roman Empire, where cultiva-
tion for food and medicinal utilisation started probably at the same time in all
provinces. After the Roman period poppy cultivation continued both in Europe
and Asia. However, opium became the main product in Asia, while poppy seed
and oil were utilised in Europe.
The opium production in Southeast Asia (Golden Triangle), West Asia
(Golden Crescent) and other territories is still going on using traditional meth-
ods. The production is regulated by local consumption, market possibilities and
political considerations. A political reason for opium production was obvious
during the ‘opium-war’ between Great Britain and China (1838–1842). At
present the policy of international organisations (WHO, FAO, UNODC) is
oriented towards gaining control of opium production in order to reduce its
illicit trade and consumption.
In Europe, a large amount of poppy oil had been produced for food use at
the end of the 18th and in the first half of the 19th century in France (Provance,
Alsace) and Germany. Later, the importance of poppy oil declined, and indus-
trial application remained the main branch of poppy utilization. In the 19th
century the manufacture of morphine started in small pharmaceutical compa-
nies using opium imported from Turkey and Persia. About 45% of the world’s
morphine production is still based on this traditional source (Bryant 1988), and
800–100 tonnes of licit Indian opium are processed annually.
J. Bernáth (*)
Department of Medicinal and Aromatic Plants, Corvinus University of Budapest,
Budapest, Hungary
e-mail: jeno.bernath@uni-corvinus.hu

450 J. Bernáth and É. Németh
In the first half of the 20th century a remarkable advance was achieved
for extracting morphine and related compounds from poppy straw. The
invention of the Hungarian pharmacist Kabay in 1928 opened a new
perspective for the industrial utilization of poppy (Bernáth 1998). Based
on his method the alkaloids, especially morphine had been extracted from
capsules. Before, straw was known as a waste, which had to be separated
from the seed as the final step of commercial poppy cultivation. Using the
new method a high seed quality crop and a valuable raw material for
pharmaceutical industry were available at the same time. The invention
resulted a continuous increase of the poppy cultivation area in Europe
and most recently in Australia. Considering the licit production area and
quantity of harvested poppy straw and seed, the main European producers
are Hungary, Spain, France, Romania, Slovakia, the Czech Republic and
Poland (INCB 2000). Germany, Austria and the Netherlands also produce
smaller amounts of poppy seed, almost exclusively for local utilisation. In
Turkey the annual poppy production is about 70,000 ha (INCB 2000). In
the late 1960s, Australia appeared as a new poppy producer; the cultivation
area located in Tasmania increased up to 21,000 ha till 2001.
In spite of the great pharmaceutical and nutritional value of poppy, a
large amount of the crop is cultivated for illicit purposes. According to data
of Bryant (1988) around 40,000 tonnes of opium is produced world-wide,
and only 5% of it is used as a raw material of industrial production or
source of seed. This contradictory situation was recognised even at the
beginning of the 20th century, and international efforts were initiated
against drug abuse and illicit traffic; as a result of the international harmo-
nisation a special board was brought into existence for checking and advis-
ing global tendencies (Bernáth 1998). In European countries, parallel to
administrative regulations, a new strategy was adopted by governments
interested in large scale cultivation of poppy. Utilising the huge biodiversity
of the species, selection had been intensified into three main directions
(Bernáth and Németh 1999):
– Producing cultivars with high alkaloid content (1.5–2.5% morphine or
thebaine), which are reserved for pharmaceutical and large-scale seed
production, only. This genetic pool can be cultivated in strictly controlled
agricultural areas.
– Selection of cultivars with low morphine content (less than 0.1%).
These cultivars are suggested for seed/oil production in any agricul-
tural region.
– Ornamental cultivars showing special morphological characteristics of
flower and capsule. This type of cultivar can be grown without restriction
because of low morphine content and small cultivation area.
Consequently, the appearance of new types of cultivars required a revision of
cultivar registration and DUS testing, for which new evaluation procedures had
to be developed (Köck et al. 2001).
15 Poppy 451
Although poppy is one of the few species utilised or even cultivated in

prehistoric time, its origin is not yet fully clear. Until the 1930s Papaver
setigerum D.C. was considered as the ancestor of poppy, which was supported
by the finding of fossils. It became clear that poppy was known by the cavemen
living in the territory of Spain, France, Germany and Hungary 4–5 thousand
years BC. This proves that some form of poppy was widely know and utilised in
ancient time (Tétényi 1997). However, based on cytogenetic investigations
(Hrishi 1960), large differences between P. setigerum and the cultivated poppy
were found questioning the P. segiterum origin. It now appears acceptable that
the gene-centre of cultivated poppy is Central Asia, especially in the territories
of Iran and Afghanistan.
On the basis of written historical records the gene center of poppy had to be
located in western Asia (Simmonds 1976). This is supported by early historical
records, which show ritual and therapeutic-like applications of the plant in this
area. The name of the plant appeared in the classic literature such as in Odyssey
and Iliad of Homer. Kritikos and Papadaki (1967) summarizing the historical
evidence concluded that the Greeks portrayed their divinities Hypnos, Nyx and
Thanatos with poppies. The first written record dates back to the eighth century
BC. In the Corinth region a city was named Mekone (Poppy-town); the name of
the town may reflect the fact that there was an extensive cultivation of poppy, or
it was the place of first discovery of the plant. In early records the plant was
mentioned as a tool for attaining an easy and painless death. Hippocrates
(460–377 BC) was one of the first who emphasised the medical advantages of
poppy and its preparations. He mentioned that the plant was frequently used in
medicinal preparations in unripe, ripe and baked forms, and he also recognised
the nutritive property of the seeds. Herakleides (340 BC) reported the plant to
be used as a tool for euthanasia in some Greek islands. People, especially
women took poppy to shorten the time until natural death.
Dioskourides (first century AD) distinguished between several kinds of
poppy. The ‘cultivated’ or ‘garden’ poppy was used in baking bread. Two
types were known in this category, plants with elongated capsules and white
seeds, and plants forming involuted and elongated capsules with black seeds.
From the present botanical point of view, those types represent the species
Papaver somniferum. Another group named ‘flowering’ poppy showed high
hypnotic properties and may refer to the species Papaver hybridum. The ‘wild’
poppy group may be equivalent to the species Papaver orientale. Later, Pliny
mentioned an ‘intermediate’ type between the ‘wild’ and ‘cultivated’ poppy,
which may be Papaver rhoeas.
There is evidence that poppy was cultivated by Sumerians, Babylonians and
Assyrians at about 3–6 thousand years BC. On the clay tables of Sumerians the
production method of poppy juice was detailed, which was collected very early
in the morning; it was called ‘gil’ and was used for curing. Poppy was also
known in early ancient Egypt, to where it was introduced from abroad,

particularly from Greece and Babylon.
The name and preparations of poppy appear both in the Bible and in the
Talmud. Probably the plant head ‘rosch’ refers to the capsule of Papaver
setigerum.
The exact time of the introduction of poppy into India is under discussion.
Probably it had been introduced at the time of the invasion of Alexander the
Great (fourth century BC). The Persians carried poppy for the needs of their
army. Later, no evidence of poppy application in India is existing till the seventh
century AD. However, it is hard to believe that opium was unknown by the
Indian physicians till that time.
The phylogenetic origin of opium poppy was approached from different
points of view. Some botanists (De Candolle 1883; Fedde 1909; Soó 1968;
Hammer and Fritsch 1977) assumed that opium poppy originated from P.
setigerum. Another hypothesis was that P. glaucum is the ancestor of the
opium poppy (Rothmaler 1949), and by others, a parallel evolution of P.
somniferum and P. aculeatum was supposed (Reckin 1971). A triploid hybrid
origin of P. somniferum (2n = 22) was assumed as well, which was supported by
morphological evidence derived from interspecific crosses (Kadereit 1986). The
crosses of P. glaucum (2n = 14) with P. gracile (2n = 14) produced progeny
with capsules similar to P. somniferum subsp. setigerum, but leaves and petals
similar to subsp. somniferum. Nevertheless, the triploid origin of P. somniferum
was also supported by cytogenetic studies. Thus, an F1 hybrid of subsp.
setigerum (2n = 44) with subsp. somniferum (2n = 22) has 2n = 22 and when
selfed, the F2 descendants were mostly 2n = 28, regaining the original basic
number of the genus Papaver (x = 7).
At present, the classification of Vesselovskaya (1975) seems to be widely
accepted. Her system is made up of three levels: the subspecies based on origin
(geography), morphology (height and branching of axis, shape of capsule) and
physiology (life cycle). However, none of these classifications dealt with
differences in alkaloid biosynthesis. An infraspecific classification of opium
poppy was established by Tétényi (1963) based on the diversity of alkaloid
synthesis and accumulation. Chemoconvars ‘Morphinan’ and ‘Isoquinoline’ as
well as their chemoprovars were distinguished. This classification was
developed taking into consideration the two demethylation pathways and the
response to photoperiod (Tétényi 1989).
15.3 Genetic Resources and Varietal Groups
In the first half of the 20th century, cultivation was mainly based on traditional
populations, and selected poppy varieties were scarcely known. In Germany,
Heeger (1956) mentions five registered cultivars, while he emphasizes the
dominance of landraces without selection in other countries. In Hungary, the
15 Poppy 453
science-based breeding had started around 1930 (Köck et al. 2001). Today, the
varietal background of the production shows characteristic differences among
cultivation areas of the world:
– In areas of highly developed industrial production (West Europe,
Australia), patented strains are used without cultivar registration by the
varietal authority. Because of industrial interest, detailed information on
them is almost fully missing. These materials are homogenous, developed
by different breeding methods and selected for special production
characteristics.
– In other countries (Central Europe) the selected cultivars are registered by
the national variety offices, similar to the varieties of other agricultural
crops. The registration authorities investigate candidates according to the
valid DUS guideline for Papaver somniferum. The official European
Union cultivar-trials for poppy are performed in Hungary. Seed and
basic information of these cultivars are available. However, in case of
industrial varieties, the propagation material is practically distributed
only by the processing factory to their agricultural producers. For the
breeders’ interest the best varieties may also be patented.
The registered varieties present in the European variety list (CPVO) are
shown in Table 15.1 based on their primary utilization. Industrial cultivars of
high alkaloid content are used for extraction and processing of pharmaceuti-
cals. Therefore, until recently, the prime goal of breeding was the increase of
alkaloid levels. During the last decades, the morphine content was increased
from 2–2.5ø (‘Ferto di zárttokú’ 1952) up to 18–20ø (‘Minoán’ 2005). The
culinary varieties are primarily used by households and in food processing,
Table 15.1 Poppy cultivars (Papaver somniferum L.) registered in European Union
(Anonymus 2007c)
Industrial (capsule) Double use (capsule and seed) Culinary (seed)
‘A- 1’ – Hungary ‘Bergam’ – Slovakia ‘Albin’ – Slovakia
‘Alfa’ – Hungary ‘Edel-Weiss’ – Austria ‘Agat’ – Poland
‘Botond’ – Hungary ‘Gerlach’ – Slovakia ‘Albakomp’ – Hungary
‘Buddha’ – Hungary ‘Kék Duna’ – Hungary ‘Ametiszt’ – Hungary
‘Csiki kék’ – Hungary ‘Major’ – Slovakia ‘Aristo’ – Austria
‘Extaz’ – Romania ‘Malsar’ – Slovakia ‘Florian’ – Austria
‘Evelin’ – Hungary ‘Marathon’ – Slovakia ‘Kozmosz’ – Hungary
‘Kék Gemona’ – Hungary ‘Marianne’ – Netherland ‘Josef’ – Austria
‘Lazur’ – Poland ‘Sokol’ – Czech Rep. ‘Michalko’ – Poland
‘Medea’ – Hungary ‘Opal’ – Slovakia ‘Mieszko’ – Poland
‘Minoan’ – Hungary ‘Parmo’ – Denmark ‘Przemko’ – Poland
‘Monaco’ – Hungary ‘Rubin’ – Poland ‘Zeno’ – Austria
‘Nigra’ – Hungary ‘Rosemarie’ – Netherland ‘Zeno 2002’ – Austria
‘Riesenmohn’ – Germany ‘Zeta’ – Austria
‘Tebona’ – Hungary
where either the seed or the oil extracted from the seed is utilized. To date, no
varieties have been developed for an increased seed oil content or for a
particular fatty acid composition.
The basic element in regulation of poppy cultivation and avoiding drug
abuse seems to be the use of proper cultivars. The production of morphine
rich cultivars is permitted exclusively under strict control (UN Agreement,
signed in 1988). On the other side, a minimum value or traces of morphine in
the capsules create the basis for uncontrolled cultivation. However, no uniform
limits between high and low alkaloid or morphine content cultivars have been
established yet. In Germany 0.01% maximal morphine value is allowed in dry
capsules; in Hungary this limit will be 0.2% from 2010 on (Németh and Bernáth
2007), whereas in the majority of countries no exact distinction level is existing.
For this reason, both industrial and culinary variety groups include a wide
range of alkaloid contents. For the industrial use we can find cultivars of
medium level (0.2–0.8%), e.g. ‘Edel-weiss’, but also ones of maximum alkaloid
level (above 1.5%), e.g. ‘Botond’. The case is similar for the culinary varieties:
only ‘Przemko’, ‘Mieszko’, ‘Mihalko’ and ‘Ametiszt’ have practically
morphine-free capsules (alkaloid level below 0.1%), whereas others exhibit
different accumulation potentials of up to 0.4–0.5% (Table 15.1). Traditional
cultivars usually exhibit a larger variability both in morphology and alkaloids,
whereas varieties released during the last 10–20 years are phenotypically more
uniform and stable. The most recent Hungarian varieties show a clear
separation between either industrial or dietary utilisation with respect to their
morphine content (Fig. 15.1).
Fig. 15.1 Separation of culinary and industrial cultivars based on the morphine content of
capsules (Németh and Bernáth 2007)
15 Poppy 455
In several European and Asian regions, the majority of cultivated materials

are landraces even today, i.e. populations without any systematic breeding.
They are well adapted to the local conditions, but usually are far from fulfilling
the requirements of intensive agriculture. In Romania, a great diversity was
found in the investigated morphological characteristics of local poppy
populations (Handrea 1996). In Turkey, the seed market offers populations
characterised by flowers of mixed colour and shape (Anonymous 2007a).
Indian landraces consist of 20–25 basic types, which have given rise to a wide
diversity by intermixing and hybridisation during long years of cultivation (Singh et
al. 1997). Representative cultivars for this region are ‘Telia-1’, ‘Bhakua’, ‘Aphuri’,
‘Kantia pink’, ‘Galania’, ‘Mandraj’ or ‘Kasuha’. Differences include plant height,
flower colour, capsule shape, morphine content and vegetation length. An inten-
sive development of genetic resources is carried out in producer countries at
present (Balci et al. 2007; Singh et al. 2003), but local populations and strains
may assure a wide and prosperous genetic basis for developing new cultivars.

The following are the major goals of current poppy breeding which are
independent of the type of utilisation:
Increase of yield had always been a primary aim of breeding. Poppy yield
includes the seeds, capsules and alkaloids in Europe and Australia, latex and
opium in India. The production of active materials is based on dry matter yield
and the accumulation level of active materials such as alkaloids or seed oil
(Bernáth 1986). However, dry matter production is a complex composed of
morphological (e.g. number and size of capsules) or physiological features
(e.g. length of vegetation period, floral biological properties, nutrient uptake
and utilisation, stem stability). Thus, improving these characteristics by
breeding indirectly rises the yield level and profitability of the crop (Singh
et al. 1997; Németh 2002).
Morphological homogeneity is considered a basic requirement for variety
registration. Previously, colour of flowers and seeds were the main features
(Mórász 1979), whereas today homogeneity of several other characteristics
needs to be assured according to the UPOV guidelines and examination
methods (Anonymous 2007b).
The significance of resistance breeding has increased during the last 20 years
connected with intensified poppy cultivation. Today, efforts for the establish-
ment of genotypes resistant against diseases such as downy mildew (Peronos-
pora arborescens) (Singh et al. 2003), damping off (Pythium dissotocum), poppy
mosaic virus (PoMo-1) (Sattar et al. 1995) and others are carried out mainly in
India. In Europe, successful selection against poppy stem rot has been practised
(Hörömpöli 1998). Although none of the poppy accessions tested till now
proved to be completely resistant against all diseases, there are wide differences
in the disease reaction of genotypes, which provides a considerable genetical

pool for resistance breeding.
Recently, resistance against abiotic factors seems to gain more attention.
Although spring sown poppy can be grown under a wide range of ecological
conditions, frost tolerant genotypes might have an economical significance
especially in Central Europe. Poppy sown in autumn and overwintering at
4–6 leaf stage usually has a superior potential compared to a spring sown
crop. It is based on some advantageous features: higher yields, earlier ripening,
avoiding the mass appearence of weevils (Ceutorrhynchus spp.) even at reduced
plant protection. However, an overwintering production presumes frost
tolerance. Therefore breeding of autumn sown ‘winter’ type varieties appears
important in poppy breeding for Central Europe (Dobos and Vetter 1997). The
respective populations have been destined primarily for culinary purposes, but
recently development of high alkaloid winter poppy types for the pharmaceu-
tical industry has been started as well (Németh and Bernáth 2007).
Further goals of poppy breeding for special characteristics are dependent of
the utilisation area:
The alkaloid content of capsules has been the key feature of poppy for
pharmaceutical purposes in Europe and Australia; since the mid of the last
century, mainly the level of morphine has been in focus. Increase of accumula-
tion to at least 1% and later 2% or even higher were achieved by selection. Since
the last 1–2 decades, a further growth of alkaloid accumulation can only be
assured through advanced breeding methods such as deliberate hybridization
or genetic transformation (Levy and Milo 1998). Through development and
broadening of the profile of pharmaceutical industries in the 1980s, new
demands appeared for cultivars accumulating other alkaloids such as narcotine,
noscapine, codeine or thebaine (Balci et al. 2007; Bernáth and Németh 1999).
Hybridization or mutation induction were applied in breeding for these new
demands (Bernáth and Németh 2005; Millgate et al. 2004). In India, poppy
breeding has always been focused on the increase of latex yield and its morphine
content. Recently, the development of cultivars morphine-rich in straw has
been started as well (Singh et al. 1997).
The lack of alkaloids is of high significance for safe seed consumption by the
confectionary industry, bakeries and households (Anonymous 2006). Poppy
cultivars accumulating minimal levels of alkaloids might be produced, pro-
cessed, transported and sold without severe restrictions and control all over
the world. The need for such cultivars for avoiding illicit use of capsules had
been recognized almost 50 years ago by Kopp et al. (1961). Development of
cultivars low in total alkaloids seems to be of high importance in countries such
as Austria, Slovakia, Hungary or Poland, where poppy seeds are a widespread
and popular dietary component. Monitoring of the fields could be accom-
plished easily if the ‘alkaloidless’ (alkaloid content below 0.1%) character is
linked to a simply observable marker trait such as flower colour, shape of leaves
or capsules etc. (Liersch et al. 1996).
15 Poppy 457
Seed colour used to be an additional breeding goal for the dietary utilisation;
poppy seeds have a higher appreciation at the market if they are dark blue. The
colour of the seeds is variable depending on pigments and the crystalline layer
below the outer epidermis (Petri and Mihalik 1998). Beside the blue coloured
cultivars, some varieties had been selected especially for white seed colour,
advised for substitution of walnut in bakery products, e.g. ‘Albin’ (Anonymous
2005) or ‘KP-Albakomp’ (Németh 2002).
Increasing the oil content as the main seed component is less frequently
mentioned as a breeding goal. Recently, the possibilities of selection for high
oil yield and for strains rich in linoleic, palmitic or oleic acid, or containing
palmitic, oleic and linoleic acids in about equal amounts had been discussed
(Bajpai et al. 1999).
15.5.1 Biological and Genetic Characteristics
Poppy is primarly an autogamous species, fertilisation occurs mostly before

opening of the flowers (Heltmann and Silva 1978). However, allogamy is also
occurring depending on variety, growing site, weather conditions, colour of the
flowers and waxyness of stigma (Bhandari 1990; Patra et al. 1992). According to
Morice and Louarn (1971), allogamy may reach 15–40% in case of European
varieties, while Khanna and Shukla (1983) describe 0–70% out-crossing in India.
The inheritance of major characteristics of poppy (Table 15.2) has been
summarized by Németh (2002). Plant height and branching of stem are
dominantly inherited traits, in some crosses over-dominance was found
(e.g. Tétényi et al. 1961).
The development of ‘double petal’ flowers as result of a modification of
anthers was described either as a recessive character (Levy and Milo 1998) or as
an effect of the multiallelic locus Pl (Belyaeva 1988). Monogenic inheritance
with multiple allelism was also found to determine the colour of petals (Bhan-
dari 1989); genotypes with irregular flowers or coloured petals have ornamental
decoration value and may be useful as marker traits for phytochemical
characteristics.
The leaves of poppy show a wide variation from the lacerate, pinnatisect to
doubly serrated. Sharma et al. (1991) suggest a digenic inheritance, where the
homozygous recessive individuals (lfr1 lfr1 lfr2 lfr2) exhibit the normal (lacerate)
leaf shape and gene-dose effects produce transition forms. Downy mildew
resistance appears to be a recessive trait most likely determined by several
genes (Kandalkar et al. 1995). Multilocus regulation is also supposed for the
colour of seeds which may be white, yellowish, greyish, brown, pink and blue at
different darkness. Based on crossing experiments, Leake and Pershad (1920)
cited in Verma et al. (1999) assumed three genes playing a role in the regulation
Table 15.2 Overview on the inheritance of major characters in poppy (Németh 2002)
Character Inheritance References
Plant height Dominance, Levy and Milo (l998)
overdominance
Heterosis Sharma et al. (1997); Kálmán-Pál et al.
(1987);
Singh et al. (1999)
Negative heterosis Singh et al. (1995)
Dominance/additive gene Shukla et al. (1999)
action
Maternal effect Khanna and Gupta (1989)
Lacerate leaf Digenic, recessive Sharma et al. (1991)
Double petal Monogenic, recessive Levy and Milo (1998)
Polyallelism Belyaeva (1988)
Divided petal Monogenic, dominant Belyaeva (1988)
Unusual stamina Monogenic, partial Belyaeva (1988)
dominance
Petal colour Monogenic, polyallelism Bhandari (1989)
Capsule length Heterosis Singh et al. (1999)
Capsule size (big Monogenic, dominant Patra et al. (1992)
capsule)
Number of capsules Heterosis Sharma et al. (1988); Sharma et al.
(1997)
Seed yield Polygenic Levy and Milo (1998); Kálmán-Pál
et al. (1987);
Heterosis Sharma et al. (1997); Singh et al. (1999)
Total alkaloid Monogenic, recessive Straka et al. (1993); Nyman and Hall
content (1976)
Polygenic Nothnagel et al. (1996)
Morphine content Intermediate Morice and Louarn (1971)
Heterosis Kálmán-Pál et al. (1987)
Negative heterosis Lal and Sharma (1995)
Overdominance Kaicker (1985)
Narcotine content Heterosis Kálmán-Pál et al. (1987)
Negative heterosis Lai and Sharma (1995)
Codeine content Heterosis Kálmán-Pál et al. (1987);
Negative heterosis Lal and Sharma (1995)
Polygenic Tóthné-Lökös et al. (1997)
Thebaine content Negative heterosis Lal and Sharma (1995)
Papaverine content Additive x dominant gene Shukla et al. (1999)
action
Latex yield Non-additive effects Shukla et al. (1997)
Heterosis Lal and Sharma (1995)
Peronospora Recessive, polygenic Kandalkar et al. (1995)
resistance.
of seed colour; however, the action of these genes on pigment content of the
parenchymatous layer only or also on the construction of the crystal cell layer,
which contributes to the colour of seeds has not yet been established.
15 Poppy 459
Heterosis and non-additive gene action may play important roles in poppy
breeding. Stronger growth of hybrids compared with the parents was described
by several authors (Dános 1965; Kálmán-Pál et al. 1987; Sharma et al. 1997). The
diameter and mass of the capsules showed a considerable heterosis (22–53%) in
many studies. As for the seed yield, the effect of heterosis may reach an even
higher level (up to 167%), while the yield of opium may increase in hybrids by
44–50% (Levy and Milo 1998; Kálmán-Pál et al. 1987; Singh et al. 1999).
A basic difference exists between genotypes accumulating alkaloids and the
ones unable to produce alkaloids in an extractable amount. According to
Nyman and Hall (1974) the extremely low level of alkaloids is due to one or
two recessive gene loci. Recent molecular-biochemical research revealed that
the first step of alkaloid accumulation is the transformation of tyrosine into
(S)-norcoclaurine. Overexpressing or silencing of tyrosine decarboxylase
enzyme (TyDC) activity may basically determine the alkaloid level of the
plant (Psenák 1998). It appears, that this enzyme is also active against dopa
(dihydroxyphenylalanine), and the TyDC/DODC gene families consist of at
least 16 genes which are organ and tissue specific (Facchini et al. 1998). Another
less studied aspect of low alkaloid content is the anatomical constitution of the
plant. Capsules of Swedish cultivar ‘Soma’ or Indian cultivar ‘Sujata’ are
almost free of alkaloids due to its underdeveloped lactiferous vessels (Straka
et al. 1993; Sharma et al. 1999), which might be inherited monogenically
through a recessive allele. With respect to the spectrum and level of different
alkaloids accumulated, there is still a lack of knowledge about the enzymes
participating in these processes. According to the majority of studies
dominance variance proved to be outstanding for morphine content. Recent
molecular genetic results demonstrate that the biosynthesis of morphinane
alkaloids is a complex process involving enzymatic and substrate feed back
reactions beside transcriptional processes (Allen et al. 2004).
Studies on the heritability of different characters have also been summarized
in Németh (2002). While relatively high heritability (h2 = 0.7–0.8) was found
for important agronomic traits (size and number of capsules, seed yield), the
heritability is lower (h2 = 0.1–0.2) in case of alkaloid accumulation.
Nevertheless, significant genetic advance was achieved in selection for alkaloid
concentrations (Bernáth and Németh 1999).
Genetic correlation between characters, particularly between alkaloids and
other traits, may be important in selection. The yields of latex, seed and oil are
positively correlated with each other (Sethi et al. 1990; Singh et al. 1995).
Heltmann and Silva (1978) describe a positive correlation of morphine content
with the number of capsules, stigmatic rays and height of plants, whereas other
studies deny any connection between capsule characteristics and morphine
(Ghiorghita et al. 1990). More likely, these connections depend on the genetic
material used and are also modified by environmental conditions (Kálmán-Pál
et al. 1989). For seed yield, Shukla et al. (2003) found the strongest correlation
with capsule mass, but also positive correlations with plant height, capsule size
and stem diameter.
In the presence of sufficient genetic variability of a starting population, selec-

tion from still existing landraces with wide distribution and high degree of
adaptability may be efficient (Handrea 1996). In poppy, selection is mostly
pactised as individual plant selection with progeny testing, which is assured by
its good self-pollination ability and seed production. However, isolation of
flowers is necessary because of the possibility of allogamous fertilisation.
The efficacy of selection for alkaloid content may be improved by
environmental selection pressure. Under low temperature and poor light
conditions narcotine and codeine can not be detected or are expressed at a low
level. Selection under such conditions could help in identifying strains with high
genetic potential for accumulation of these alkaloids (Bernáth et al. 1988). Selection
under provocative conditions is also used in resistance breeding (Hörömpöli 1998).
Hybridisation had been used in poppy breeding since the 1940s. Both intra-
and interspecific crosses of poppy may result in valuable new breeding material.
Emasculation of flowers is carried out, when pollen grains are not mature and
self-pollination has not yet occurred; after removing the stamina, isolation of
stigma is necessary, and at the same time or during the following days pollina-
tion can be done. The effect of a male parent may appear already in the tissues
of the mother plant. Bernáth and Németh (2003) proved metaxenia for the
alkaloid content of the capsules. When an alkaloid free cultivar was pollinated
by another one rich in morphine, the morphine content of capsules of the
maternal parent increased by 0.9–7.5 mg/g.
The pedigree method of selection is most widely applied. According to Levy and
Milo (1998), pedigree breeding was efficient in combining and fixing plant char-
acteristics such as capsule yield, increased opium and seed yield as well as lodging
resistance. Selection and stabilisation of desired genotypes is usually achieved in 3–4
generations. When crossing genetically distant cultivars, evaluation of the early
progenies may be difficult due to the appearance of heterosis (Dobos and Vetter
1997). Significant reciprocal effects were also found in particular crosses. The
maternal effect was most obvious in crosses between low and high alkaloid cultivars
or cultivars of different alkaloid types (Bernáth and Németh 2005).
Backcrossing is practised after interspecific hybridization for elimination of
wild-type properties and after mutation induction treatments. Shukla et al. (1999)
proposed the development of high papaverine genotypes through backcrossing.
Hybrid breeding can be applied in poppy because both self- and cross-pollina-
tion is easily possible, and the seed propagation rate is high. Kaicker (1985)
described, that heterosis effects can be utilized even for two generations because
of mainly non-additive genetic effects for economically important characteristics
(e.g. opium and seed yield). In contrast, Singh et al. (1999) found considerable
inbreeding depression for six characters (e.g. seed yield) already in the F2 genera-
tion. Selection of appropriate parents for hybrid development is carried out after
testing for combining ability in diallel trials and improving genotypes by recurrent
15 Poppy 461
or reciprocal recurrent selection (Heltmann and Silva 1978). Hybrid seed produc-
tion has to be carried out by hand-pollination which limits seed production
(Mórász 1979), as no source of male sterility is available till now. Although hybrids
are mentioned occasionally (Sharma et al. 1997), no hybrid cultivars are produced
at a commercial scale because of the hudge cost of seed production. Alternatively,
development of synthetic varieties is practised more frequently and is considered
the most suitable method in poppy breeding (Khanna and Shukla 1989). Synthetic
varieties may keep up with F1 hybrids in production capacity; besides, productivity
does not decrease radically in the following generations and seed production cost is
much lower.
Polyploid forms of poppy exhibited an increased morphine level and higher
capsule numbers (Andreev 1963 cited in Levy and Milo 1998; Chauhan and
Patra 1993). In Hungary, high expectations were held for polyploids (Kiskériné
et al. 1977 cited in Németh 2002), but no practical results were achieved due to
the decrease of seed production in tetraploid poppy (from full sterility to 7.0%
fertility compared to diploids).
Characteristics such as male sterility, lack of opium production, increase in
morphine accumulation, multiplication of capsule number, dwarf growth and
early flowering were induced through either chemical mutagenesis or irradia-
tion (Khanna and Singh 1975 cited in Levy and Milo 1998; Nigam et al. 1990).
Shifting of biosynthetic pathways into a desired direction seems to be the most
useful application of mutagenesis. As an example, non-narcotic (alkaloid-free
and opiumless) poppy with high seed and oil yield was developed through
mutagenic treatments using gamma rays (100–800 Gy) and ethyl methane
sulphonate (EMS 0.4%) (Sharma et al. 1999). Mutants were also found which
accumulate thebaine and oripavine but do not complete the biosynthesis into
codeine and morphine (Millgate et al. 2004); others accumulate high levels of
noscapine beside morphinanes (Ziegler and Kutchan 2005).
Isoenzyme markers may be applicable for cultivar identification, as demon-

strated by Margl et al. (2001) using GOT, 6-PGDH, ACP, DIA, GDH, LAP,
MDH and SAD enzyme systems which revealed 1–9 alleles in a total of 12
poppy genotypes. A genetic linkage map containing 87 markers in 16 linkage
groups was constructed from a segregating population (Straka and Nothnagel
2002) and is intended for mapping morphine content loci.
Various studies have been carried out in order to identify and isolate the
enzymes being involved in alkaloid formation of poppy. Over 30 enzymes
participating in biosynthesis of benzylisoquinoline alkaloids have been isolated
from cell cultures and are now characterised and partly purified. For many of
the enzymes identified, the involvement of multiple cell types is suggested in
alkaloid biosynthesis. As an example, while berberine bridge enzyme is
localized to parenchyma cells of the root cortex, O-methyltransferases are
found in the pericycle, and – typically – codeinon reductase is only found in

organs were laticifers are present (Weid et al. 2004).
Molecular cloning of pathway genes has been successful since the mid 1990s
and respective cDNAs are known (e.g. Ounaroon et al. 2005; Ziegler
et al. 2006). One of the first genes identified from poppy was that encoding
tyrosine/3,4-dihydroxyphenylalanine decarboxylase (Facchini et al. 1998).
Recently Ziegler and Kutchan (2005) constructed a specific cDNA library for
identification of sequences responsible for morphinane biosynthesis. From over
thousand unique sequences created and used for gene expression analysis, 27
sequences showed highly different expression when comparing poppy strains
(P. somniferum and P. bracteatum) either containing morphine or noscapine.
Agrobacterium tumefaciens-mediated genetic transformation can be
achieved in hypocotyl derived suspension cultures of poppy using antibiotic
or herbicide resistance markers for effective selection of transgenic cells and
plant regeneration through somatic embryogenesis (Nessler 1998). Chitty et al.
(2003) reported a high rate of somatic embryogenesis in the creation of trans-
genic plants; measuring outcrossing rates they concluded that a distance of
above 2.5 m between transgenic and neighbouring plants was sufficient for
avoiding outcrosses. Several practical examples in genetic transformation of
poppy have been achieved in recent years. Frick et al. (2004) transformed the
berberine bridge enzyme cDNA into seedling explants of an industrial elite line.
The selfed progenies of the regenerated plants showed an altered alkaloid
profile which was heritable. Larkin et al. (2007) enhanced the activity of
codeinone reductase enzyme thus increasing morphinan alkaloids (by up to
28% in the best transformants) in transgenic whole plants as a consequence of
over-expression of codeinone reductase. More recently, genetic manipulation of
the gene regulating (S)—N—methycoclaurine -30 hydroxylase resulted in sig-
nificant changes of alkaloid level in latex. Overexpression of the gene induced a
450% increase of the total alkaloid level, while silencing of it by antisence
cDNA caused a reduction of up to 84% (Frick et al. 2007).
Breeding of poppy has resulted in advanced cultivars in Austria, Australia,

Czech Republic, Germany, Hungary, Poland and Slovakia. Beside the variety
list of the European Union (Table 15.1), various other cultivars and strains exist
representing local selections or industrially utilized strains, especially in India.
15.7.1 Industrial Cultivars for Alkaloid Production

Widely grown cultivars already in the middle of the last century were e.g.
‘Mahndorfer’, ‘Strubes Blauer’, ‘Eckendorfer Blausamiger’ in Germany,
15 Poppy 463
‘Kompolti M’, ‘BC-2’, ‘Kék Duna’ in Hungary and ‘Reading’ in England.

During the last decades, new industrial cultivars had been developed with
continuously increasing alkaloid contents of up to 2–3%, capsule yields of
over 1,000 kg/ha and adaptation to intensive cultivation. In Hungary, cultivars
accumulating other alkaloids beside morphine, e.g. ‘Kék Gemona’ (narcotine),
‘Monaco’ (codeine) or ‘Tebona’ (thebaine) are produced on large scale.
In India, high yielding cultivars are ‘Rajhans Chetak’, ‘Kirtiman’, ‘JA-16’ or
the synthetic variety ‘BROP-1’ (Singh et al. 1997); the best genotypes assure an
opium yield of 250–320 mg/plant with 15% morphine content. The cultivar
‘Pusa Selection 1-3’ provides more than 53 kg/ha opium (Kaicker 1985). Other
strains (‘MOP-539’, ‘G-38’, ‘BF-5-13’) have been developed for downy mildew
resistance.
In Australia, a population (‘Top-1 poppy’) representing a morphine less and
thebaine rich mutant has been developed, which provides precursors for highly
effective analgesiscs and for treatment of opioid addiction (Millgate et al. 2004).
15.7.2 Culinary Cultivars for Poppy Seed and Oil
Breeding of poppy started from populations cultivated for edible seeds and oil
production. Oil content of poppy seed is in the range between 40 and 55%, and
palmitic acid (C16:0, 10–12%), oleic acid (C18:1, 12–22%) and linoleic acid
(C18:2, 60–75%) mainly make up the fatty acid composition. However, seed
yield, oil content or fatty acid composition have never played a primary role in
poppy breeding. During the last decades, breeding for culinary purposes
focused on decreasing morphine content of capsules in order to avoid drug
abuse and contamination of seeds.
The Swedish low-alkaloid cultivar ‘Soma’ is the result of a spontaneous
mutation. It had been the base for developing of low-alkaloid varieties
such as the Polish variety ‘Przemko’ with a morphine content of 0.05%
and further lines even lower in morphine and with marker traits such as
laciniated and coloured flowers (Liersch et al. 1996). Subsequently, culti-
vars ‘Michalko’ and ‘Mieszko’ have been developed for free seed utilisa-
tion and cultivation, blue seed colour, yield potential of 1–1.2 t/ha and oil
content of 48–49%. From crosses between gene bank accessions followed
by pedigree selection, the Hungarian culinary cultivar ‘Ametiszt’ had been
developed which accumulates only 0.03–0.08% alkaloids in the capsules;
besides, its purple-pink petals represent a good morphological marker of
its non-narcotic characteristic (Németh et al. 2002). In India, the variety
‘Sujata’ was produced as a result of mutagenic treatment. The plant does
not exude latex on lancing and seed oil content is in the range of
50.7–53.5% (Sharma et al. 1999).
In Central Europe, winter-sown poppy cultivars may reach superior yields
compared to spring-sowing; therefore, improvement of frost tolerance is a main
breeding objective. The first winter-sown Hungarian cultivar ‘Kozmosz’ is too

high in alkaloid level (0.4–0.7%) at present to be used in free production. The
Austrian winter poppy cultivar ‘Zeno Wintermohn’ which had been developed
from a land race, out-yields spring-sown cultivars by more than 30% reaching
1,450 kg/ha of seed (Dobos 1996); improved strains have been developed from
that population recently.
Acknowledgment The Hungarian poppy research has been supported since 2000 by the
Hungarian Research Fund (OTKA, No. T32393 and K62732).
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Chapter 16
Hull-Less Oil Seed Pumpkin
Tamás Lelley, Brent Loy, and Michael Murkovic
16.1 Introduction
Pumpkin is one of the oldest domesticated crops of the Neolithic revolution

(Smith 1997; Piperno and Stothert 2003), and pumpkin seeds are consumed as a
snack in many cultures throughout the world. Since the bitter flesh of wild
Cucurbita species is usually inedible, very likely seeds were the first parts of
pumpkin eaten by humans (Whitaker and Bemis 1964).
In the volume ‘Vegetables I’ of the ‘Handbook of Plant Breeding’ two
separate chapters are dealing with ‘Pumpkin and Winter Squash’ (Ferriol and
Picó 2008) and ‘Summer Squash’ (Paris 2008), respectively. Most of the relevant
information on breeding cucurbits as vegetables is handled in those two
chapters. Hull-less oil seed pumpkin (Cucurbita pepo subsp. pepo), however, is
a specialty oil crop of Central Europe with a number of specific problems not
covered by those chapters, which justifies an in-depth treatment of the subject in
the present contribution.
16.1.1 Use of Hull-Less Pumpkin Seed As a Food Crop

The first definite proof for cucurbit cultivation in Styria, Austria was found in
the legacy of a farmer dating back to 1697, in which 54 l of pumpkin seeds were
recorded (Riegler 2004, loc.cit. Teppner 2004). Several references to pumpkin
cultivation in the region and the use of pumpkin seed oil in the early 18th
century can be found in Riegler (2004, loc. cit. Teppner 2004) and Kundegraber
(1988, loc. cit. Teppner 2000, 2004). The seeds used for oil production were
manually de-hulled (Hlubek 1860, loc. cit. Teppner 2004). Based on indirect
evidence as summarized by Teppner (2000, 2004), the approximate time of
emerging and spreading of a naked seeded, also called thin-coated or hull-less
T. Lelley (*)
Department of Agrobiotechnology Tulln, University of Natural Resources and
Applied Life Sciences Vienna (BOKU), Tulln, Austria
e-mail: tamas.lelley@boku.ac.at

470 T. Lelley et al.
mutant of Cucurbita pepo subsp. pepo in the southwestern part of the then
Austro-Hungarian Monarchy appears to be dated between 1870 and 1880. Due
to the popularity of pumpkin seeds and especially the seed oil, the advantage of
the hull-less seed type, allowing a much more efficient oil extraction, was
quickly recognized, leading to its rapid dispersal in the whole region. Essen-
tially, this mutation turned pumpkin into an oil crop. Pumpkin was grown in
Austria in 2006 on 18,151 ha with an average seed yield of 0.61 t/ha.
The first hull-less oil-pumpkin variety, ‘869 Feldkürbis’, appeared in a seed
catalog of 1915 (Teppner 2004). The first variety derived from a cross between a
hull-less oil pumpkin and a Vegetable Marrow genotype, combining the bush
growth habit of the latter with the hull-less seed type of the former, was
registered in Austria from 1955 to 1974 under the name of ‘Tschermaḱs
Ölkürbis’. Tschermak also wrote the first extensive paper on the hull-less
pumpkin (Tschermak-Seysenegg 1934). The scientific name, C. pepo subsp.
pepo, var. styriaca I. Greb., was conferred to the Styrian oil-pumpkin by
Grebenščikov (1950). At present five open pollinated and three hybrid cultivars
of oil pumpkin are registered in the Austrian variety list (AGES 2008).
In the USA Curtis (1948) first recognized the potential value of hull-less
seeded pumpkin for producing a high quality vegetable oil and for use as a
snack seed, and initiated the first breeding program at the University of Con-
necticut. However, Curtis left Connecticut before he could bring this project to
fruition. In breeding work at the University of New Hampshire, two hull-less
seeded, small-fruited squash cultivars were released, ‘Sweetnut’ in 1960 and
‘Eat-all’ in 1965, followed by the bush pumpkin, ‘Tricky Jack’ in 1969. How-
ever, the first variety of a naked seeded oil-pumpkin to receive more national
acclaim was ‘Lady Godiva’ released by the USDA in 1972. Phenotypically,
‘Lady Godiva’ shows close similarity to Styrian oil pumpkin.
16.1.2 Origin of the Hull-Less Seeded Phenotype in Pumpkin
Grebenščikov (1954) suggested a common origin in Styria for all hull-less

pumpkin genotypes around the world. The absence of any trace of lignin in
the seed coats of F2 progenies derived from crosses of hull-less genotypes from
Argentina, South Africa and China with completely hull-less strains of Styrian
oil-pumpkin substantiates the postulation of a common genetic background for
the hull-less phenotype, and that all of them were probably directly descended
from the same Austrian ancestor (Zraidi 2005). Further evidence was provided
by Stift et al. (2004) by means of a genetic distance analysis, carried out using
SSR markers and comparing Styrian cultigens with hull-less genotypes from
China, South Africa, and Argentina. The hull-less phenotype likely evolved
from the rare occurrence of a single recessive mutation event as proposed by
Grebenščikov (1954), and in Austria there would have been a strong incentive
for selection of such a phenotype given the interest in oil seed pumpkin. This
16 Hull-Less Oil Seed Pumpkin 471
would certainly have been followed by subsequent breeding efforts, perhaps

through cross breeding, to select for modifying genes which further reduced
lignification of the seed coat. Corroborating the speculation of initial appear-
ance of a single gene mutant in C. pepo is the intriguing report by Zhou Xianglin
(1987) of a hull-less genotype of Cucurbita moschata Duchesne, discovered by
the author in a landrace collection in the Shanxi province in China. The hull-less
seeds are, however, white, due to a complete lack of protochlorophyll in the
chlorenchyma layer, whereas Styrian oil pumpkin seeds are green. Successful
crossing of this genotype with zucchini and further crossing the F1 with Styrian
oil pumpkin yielded a progeny showing a clear 1:1 segregation suggesting that
these two genes may be alleles (Pachner and Lelley unpublished results), repre-
senting a classical example of Vavilov’s parallel variations (Vavilov 1920).
16.2 Nutritionally Relevant Components of Pumpkin Seeds

The main nutritionally relevant components of pumpkin seeds are protein and
oil. Albumins and globulins, in the range of 59% of the crude protein, are the
most prominent protein fractions. The oil content was evaluated by Murkovic
in different breeding lines. Breeding lines and varieties commonly used for
commercial production of seeds have an oil content of 46.9/100 g (standard
deviation 2.4/100 g, n = 184, Murkovic, unpublished results). The main fatty
acids of pumpkin seeds are palmitic acid (C 16:0), stearic acid (C 18:0), oleic
acid (C 18:1), and linoleic acid (C 18:2). These four fatty acids comprise more
than 98% of all occurring fatty acids. Linoleic acid is the most prominent,
followed by oleic acid and palmitic acid. The values are shown in detail in
Fig. 16.1. All other fatty acids are present in minor concentrations. The Chinese
variety of Cucurbita moschata, ‘Zhou’, shows a similar fatty acid distribution
(C16:0 16.6%, C18:0 7.6%, C18:1 17.3%, C18:2 56.7%). Linoleic acid belongs
to the group of essential fatty acids which is modified metabolically to
eicosanoids. A low level of eicosanoids arising from an undersupply with
linoleic acids is partly responsible for the well known deficiency symptoms of
linoleic acid (Innis 1996).
Fig. 16.1 Distribution of

fatty acid content in
pumpkin seeds (n = 400;
updated from Murkovic
et al. 1996a)
Depending on climatic conditions, the fatty acid composition is subject to

variation. When the temperature is lower during the last weeks of seed filling,
there will be a shift from oleic to linoleic acid. For example, in the year 1994 the
average temperature from July to October was 15.88C, resulting in a relative
concentration of linoleic acid of 48%, whereas in the following years the
temperature was below 15.28C and linoleic acid rose to 57% (Murkovic
et al. 1999).
Tocopherols present in the seeds are dominated by g-tocopherol
(41–620 mg/kg), the concentration of which is 5–10 times higher than the
concentration of a-tocopherol (0–91 mg/kg) (Fig. 16.2). Additionally, a sig-
nificant amount of a-and g-tocotrienols is present. In contrast to the fatty
acids, the content and distribution of tocopherols is not influenced by the
climate during the ripening period (Murkovic et al. 1996b). In addition to the
vitamin E activity and radical quenching properties, tocotrienols are known
for their potential to reduce the incidence of breast cancer (Nesaretnam et al.
2007).
Fig. 16.2 Content of a- and

g-tocopherol in pumpkin
seeds (n = 400; updated
from Murkovic et al. 1996b)
Pumpkin seeds also contain phytosterols that are dominated by D7-sterols

(Table 16.1). The amount of D5-sterols is significantly lower than in other seed
oils. Due to this unique phytosterol composition of the seed oil, the analysis of
phytosterols can be used to detect adulteration of pumpkin seed oil with other
Table 16.1 Summary of sterol and secoisolariciresinol (SECO) content in pumpkin seeds and
seed oil. Values are means of three replications with standard deviation (from Murkovic et al.
2004)
24-ethylcholest-7-enol, 24-ethylcholest-7,22- Total amount of SECO (mg/g
dienol and co-eluting sterols (mg/g FW) sterols1 (mg/g FW) FW)
Seeds 860 30 1710 80 3.8
Oil 2060 10 4030 110 n.d.
n.d.: below limit of quantification.
1
includes e.g. D7-mono- and diunsaturated sterols with 29 C-atoms, dimethylsterols, cam-
pesterol and stigmasterol.
plant oils. Mandl et al. (1999) found 1,160 mg/ml total phytosterols in pumpkin
seed oil out of which 58 mg/ml was b-sitosterol. In addition to the 5-a-reductase
inhibiting properties, phytosterols are known for their cholesterol lowering
effects (Thompson and Grundy 2005). However, the concentrations of phytos-
terols present in functional foods that are used to lower the serum cholesterol
level are much higher than in pumpkin seeds.
Carotenoids found in the pumpkin flesh and the seeds are physiologically
important, especially lutein, which may reduce risk of the development of age
related macula degeneration (AMD, Stringham and Hammond 2005). Matus
et al. (1993) found some carotenoids in the defatted seed meal, undoubtedly
originating from inner seed coat (chlorenchyma), the primary source of car-
otenoids in seeds of C. pepo (Loy, unpublished results). The main components
of the press-residue were lutein (3,30 -dihydroxy-a-carotene = (3R,30 R,60 R)-b,
e-carotene-3,30 -diol; 52.5%) and b-carotene (b,e-carotene; 10.1%). In addi-
tion to the above mentioned pigments, small quantities of violaxanthin,
luteoxanthin, auroxanthin epimers, lutein epoxide, flavoxanthin, chrysanthe-
maxanthin, 9(90 )-Z-lutein, 13(130 )-Z-lutein, 15-Z-lutein, (central-Z)-lutein,
a-cryptoxanthin, b-cryptoxanthin, and a-carotene (b,e-carotene) were identi-
fied. These carotenoids are not only found in the seeds, but also in the flesh of
the fruits (e.g. Murkovic et al. 2002; Azevedo-Meleiro and Rodriguez-Amaya
2007).
Another group of physiologically active substances are lignans, especially
secoisolariciresinol is a compound of interest. The concentration in the seeds
was determined by the group of Sontag (Murkovic et al. 2004, see Table 16.1)
being 3.8 mg/g, whereas Adlerkreutz and Mazur (1997) found a significantly
higher amount of 200 mg/g. This group also reported the presence of other
estrogens like genistein and daidzein in pumpkin seeds, but the concentrations
of these isoflavones are several orders of magnitude lower compared to
soybean products. Lariciresinol was identified by Sicilia et al. (2003) at trace
levels. Experimental evidence in animals has shown clear anti-carcinogenic
effects of pure lignans in many types of cancer. Many epidemiological results
are controversial, partly because the determinants of plasma metabolites are
very different in different countries. The source of the lignans seems to play a
role, because other factors in the food obviously participate in the protective
effects.
The seeds contain different protochlorophylls in the innermost layer of the
chlorenchyma (Teppner 2004). Chlorophylls are not formed inside the fruits,
since the complete synthesis requires a light induced reaction (Mukaida
el al. 1993). During the process of oil pressing, these pigments are extracted
into the oil providing its typical, dark green color. Since chlorophylls are known
as photosensitizers in lipid oxidation, this makes it necessary that the oil is
stored in the dark. The oil of Chinese Cucurbita moschata variety ‘Zhou’,
mentioned above, may be more resistant to photooxidation, since the seeds
do not contain these pro-oxidant chlorophyll precursors.
16.2.1 Antioxidant Activity of Pumpkin Seed Oil
Fruhwirth and co-workers developed a method that uses a fluorescence marker,

1-palmitoyl-2-(2-(4-(6-phenyl-E-1,3,5-hexatrienyl)phenyl-carbonyl) – sn-gly-
cero-3-phosphocholine (DPH-DC)). As the fluorophore is sensitive to
oxidation, a reduction of the fluorescence indicates the oxidation process. The
oxidation experiments were carried out under physiological conditions at 378C.
Using this test, the pumpkin seed oil as well as extracts from seeds with
non-polar solvent showed a very high antioxidative capacity compared to
other edible oils. The protection against oxidation was attributed partly to the
presence of tocopherols and partly to polar substances that also react with the
Folin-Ciocalteu reagent. This reagent is commonly used to determine
polyphenols (Fruhwirth et al. 2003).
In Austria pumpkin seeds are used for curing bladder and prostate
associated symptoms in traditional medicine. In modern phytotherapy the
enriched polar extracts of pumpkin seeds are used for curing benign prostate
hyperplasia (BPH). The hypothesis of the pharmaceutical action is based on the
inhibition of 5-a-reductase. The enzyme 5-a-reductase (testosterone 5-a-reduc-
tase, EC 1.3.99.5) converts testosterone to dihydrotestosterone (DHT), which is
the active male sex hormone. The enzyme is a nuclear membrane bound
NADPH-dependent d-3-ketosteroid 5-a-oxido-reductase (5-a-reductase). The
testes and adrenal glands secrete testosterone into the blood stream. Then a
considerable amount of this substance, diffusing into 5-a-reductase containing
cells, is irreversibly converted to DHT. Both testosterone and DHT produce
androgen-mediated effects; however, DHT is significantly more potent. DHT
promotes the development of prostate cells and BPH, and possibly serves as a
promoter for prostate cancer. Therapy with 5-a-reductase inhibitors such as
finasteride causes decreased serum prostate DHT levels, regression of BPH, and
a reduction of serum prostate specific antigen (PSA). PSA is a prostate
specific marker, an elevated level of which may indicate prostate cancer
(Brawley et al. 1994; Bartsch et al. 2002).
Together with other phytopharmaceuticals (Pygeum africanum – african
plum; Populus tremula – aspen; Serenoa repens – dwarf palm; Echinacea
purpurea – purple cone flower; Secale cereale – rye; Hipoxis rooperi – South
African star grass; Urtica dioica – stinging nettle; Aletrius farinose – Unicorn
root), also Cucurbita pepo was studied for its ability to relieve symptoms related
to BPH and alter 5-a-reductase activity. What is normally investigated in
these studies are parameters like modified Boyarsky symptom score,
International Prostate Symptom Score (IPSS), quality-of-life (QOL) index,
maximum urinary flow (Qmax) and postvoid residual urine volume (PVR).
Most of the studies, in which the efficiency of phytotherapeutic agents for
BPH was tested, have been carried out without controls. The difficulty of
such studies is their lack of considering a high placebo effect of 40–60%
observed in several controlled studies (Lowe and Ku 1996). Additionally,
the active principle is normally not known, with the result that the dose of
the active ingredient is also unknown, since a standardization of the extract
is not possible (Dreikorn et al. 2002). A placebo-controlled study (Bach
2000) showed that within 12 months a significant reduction of IPSS was
obtained by administering a polar pumpkin seed extract compared to the
placebo, whereas other parameters investigated (Qmax, QOL, prostate
volume, PVR) did not change.
16.2.2 Treatment of Symptomatic BPH with b-Sitosterol

A 6-month randomized double blinded trial with an 18-month follow-up has
shown that b-sitosterol improved the parameters related to BPH (modified
Boyarsky symptom score, IPSS, QOL index, Qmax and PVR). A dose 20 mg
of b-sitosterol, along with minor amounts of b-sitosterol-b-D-glucoside and
other phytosterols, was applied 3 times daily. The active ingredients in this
mixture are unknown (Berges et al. 2000). If b-sitosterol was the active ingre-
dient in this study, and this is compared with pumpkin seeds, this is equivalent
to 50 ml of oil (results from Mandl et al. 1999), or 15 g of oil, if the total amount
of phytosterols is considered (Murkovic et al. 2004). Mandl et al. (1999),
however, found significantly lower values of b-sitosterol in the pumpkin seed
oil. Thus, for an uptake of 20 mg of phytosterols a much higher amount of oil
has to be consumed.
The evidence for the efficacy of phytotherapeutic agents in the treat-
ment of symptomatic BPH is inconclusive. Although a few studies were
published which showed positive effects of pumpkin seed extract on pros-
tate symptoms, a recommendation of these products at this point still lacks
sufficient scientific support. However, since many of the medicines used in
clinical practice are derived from the plant kingdom, it is conceivable that
these agents do have beneficial effects in the treatment of BPH. The
widespread usage and demand for these agents certainly warrant further
well-designed, long-term, placebo-controlled studies (Lowe and Ku 1996).
Taking all health promoting substances into account (linoleic acid, vitamin
E, lutein, lignans, phytosterols) pumpkin seeds certainly contribute to a
healthier diet.
16.3 Genetics of the Hull-Less Seed Character
The seeds of C. pepo normally are covered by a thick, leathery, whitish to ocher
coat consisting of five cell layers (hull), of which at least three are strongly
lignified (Fig. 16.3a). The testa of the pumpkin seed is of maternal origin, thus
seeds inside the fruit should theoretically show the same seed coat genotype.
Styrian oil pumpkin seeds are characterized by a dark green color, unique for
Fig. 16.3 Hulled (a) and

hull-less (b) seeds of C. pepo
this genotype, due to a complete lack of lignification of any of the testa layers
(Fig. 16.3b). The outer testa layers collapse, resulting in a transparent layer
through which the high protochlorophyll content of the well-developed fifth
layer, chlorenchyma, comprising up to 12 cell-layers (Stuart and Loy 1983)
becomes visible. Protochlorophyll is the immediate precursor of chlorophyll.
Several studies on the seed coat character and its inheritance were carried
out in the early 1950s (Heinisch and Ruthenberg 1950; Schöniger 1950, 1952,
1955; Grebenščikov 1954; Prym von Becherer 1955). There is general agree-
ment on the existence of a major dominant gene responsible for the wild type
seed coat. In a cross involving a hulled and a completely hull-less genotype,
the seed type in 3/4 of the F2-plants is clearly hulled, 1/4 being hull-less.
Nevertheless, these hull-less seeds may show variation with respect to the
amount of lignin deposited in the testa, ranging from its complete absence
up to a clear lignification, represented by a thin layer covering the whole seed
surface. This condition will be referred to as ‘residual lignification’. To
account for this variation, different genetic interpretations were put forward,
such as the presence of a major recessive gene (Schöniger 1950; Grebenščikov
1950), a minor gene and/or modifiers (Schöniger 1952, 1955; Grebenščikov
1954; Stuart 1983), or a multigenic model (Mudra and Neumann 1952;
Teppner 2000, 2004).
Histological investigations demonstrated that the seed-coat development up
to 10 days post-anthesis is similar in both wild and hull-less seed types. It results
in five clearly distinguishable tissue layers: epidermis, hypodermis, sclerench-
yma, aerenchyma and chlorenchyma (Stuart and Loy 1983). The effect of the
mutation on reduced lignification of the sclerenchymatous and hypodermal
layers and reduced polysaccharide deposition in the epidermis becomes clearly
visible by 20 days post-anthesis. While in the hulled wild type the second, third,
and fourth tissue layers become strongly lignified, in the hull-less types due to
the lack of lignin deposition these tissue layers collapse forming a transparent
hyaline (Schöniger 1950; Stuart and Loy 1983).
Typically, Styrian oil pumpkin lacks any residual lignification. Crossing
Styrian oil pumpkin with zucchini results in a clear 3:1 segregation, 3/4 being
completely hulled and 1/4 showing variation with respect to residual lignifica-
tion. This variability extends from a complete lack of lignin to a complete
coverage of the seed surface by a thin lignified layer. Recently, an attempt
was made to clarify the genetic background of this residual lignification
(Zraidi 2005). Three oil pumpkin varieties were crossed with one crookneck
and two zucchini genotypes, respectively. Analysis of the three F2-populations
proved that the seed coat phenotype is primarily controlled by a single major
dominant gene H, as was first suggested by Schöniger (1950) and confirmed by
Grebenščikov (1954). The homozygous dominant HH and the heterozygous
genotypes Hh produce completely hulled (3/4) seed type (Type 1), while seeds of
the hh genotypes are hull-less (1/4), but with a varying expression of residual
lignification. This varying expression of the hull-less seed type was classified
following Schöniger (1950, 1955) as seed coat Type 2 (Fig. 16.4a), Type 3
(Fig. 16.4b) and Type 4 for completely hull-less seeds (Fig. 16.3b).
Fig. 16.4 Hull-less seeds of

Type 2 (a) and Type 3 (b)
Schöniger (1955) attributed this variation to the presence of a second,

incompletely dominant minor gene N, the effect of which is not discernible in
the presence of the major gene H. In homozygous dominant condition it is
responsible for the production of a thin coat, covering the whole surface of the
seed (Fig. 16.4a, Type 2). In heterozygous condition, this thin coat is partial and
develops only in parts of the seed surface usually spreading from the margin
towards the middle (Fig. 16.4b, Type 3). The recessive alleles produce comple-
tely hull-less seeds (Type 4, Fig. 16.3b). Similar results were reported by Stuart
(1983), but to account for a low occurrence of completely hull-less segregants
(Type 4) and approximately equal proportions of Type 2 and Type 3 pheno-
types, a three gene model was invoked, one major gene plus two modifiers. The
three gene model could explain the F2 segregation results, but not the testcross
results. However, it was noted that ‘Tricky Jack’and 293A, the hull-less parents
used in the inheritance study, exhibited seasonal differences in expression of the
hull-less trait, varying from Type 2 to Type 4 (Stuart 1983).
Schöniger’s hypothesis of two genes was first criticized by Grebenščikov
(1954). Not being able in his experiment to prove the presence of a second
gene for the phenotypic variation of the hull-less seeds, he accepted the sugges-
tion of the presence of a major dominant gene (H) segregating in three to one
hulled versus non-hulled types. For the varying expression of a thin coat
observed on the non-hulled seeds, however, he proposed modifiers of which the

inheritance ‘can not be explained on the base of Mendelian segregation’. The
genetics of the seed coat character was also investigated by Teppner (2000)
using a C. pepo genotype originating from Georgia. This genotype he later
classified as C. pepo var. georgica with a seed coat which is thinner than in the
wild type but thick enough to hide the chlorenchyma and to possess a lignified
margin. This type was named semi-thin by Teppner. He crossed this genotype
with hull-less Styrian oil-pumpkin and found that the semi-thin character of the
Georgian genotype was dominant. The F2-generation segregated in thick
coated, semi-thick, semi-thin and thin coated types. In his final conclusion
Teppner suggests that to account for all the variation in seed coat phenotypes
6–12 genes could be responsible (Teppner 2000).
The results of Zraidi (2005) clearly confirm the existence of a major com-
pletely dominant gene conferring the hulled wild type character to C. pepo. If
this gene is named H (Schöniger 1950), the hull-less type has the genotype hh. In
the new gene-list of Paris and Brown (2005), both genes h and n are described
conferring the hull-less character to seeds of C. pepo (hh) (Schöniger 1950) and
of C. moschata (nn) in which a hull-less seeded genotype was discovered by
Zhou (1987). Crossing this hull-less genotype, No. 6518, with a hulled one he
found a clear 3:1 segregation of hulled versus hull-less types. This line is now
named ‘Zhou’. As described earlier, results of the crossing experiment of
Pachner and Lelley (unpublished results) strongly suggest that this is the same
gene as in C. pepo. Hence, for priority reasons, this gene should be named
h instead of n as proposed by Zhou (1987), especially because for the residual
lignification of the hull-less seeds Schöniger (1950) postulated a minor incom-
pletely dominant gene, which she named N or n, and the existence of that gene is
not yet disproved.
At the histological level, as described in Zraidi (2005) and Zraidi et al. (2003)
the thick coat covering the seeds of the hulled types was due to the strong
lignification of the three testa cell layers: hypodermis, sclerenchyma and aer-
enchyma. If on the seed surface of the hull-less segregants a continuous thin
coat was observed (Type 2, Fig. 16.4a), this was always accompanied by a
continuous but weakly lignified sclerenchyma cell layer in the histological
picture (Fig. 16.5b) as was already indicated by Schöniger (1950). According
to Zraidi (2005), it is possible that the dominant major gene H in the hulled type
is responsible for the lignification of all but the chlorenchyma and epidermal
cells, while the incompletely dominant gene N is responsible exclusively for the
lignification of the sclerenchyma layer in hull-less Type 2 and Type 3 seeds.
Consequently, completely hull-less seeds (Type 4) result from the homozygous
recessive state of both genes (hhnn). It is worth mentioning that the histological
appearance of hull-less C. moschata is similar to that of hull-less C. pepo, Type 4.
The seeds are, however, white because of the absence of protochlorophyll in the
chlorenchyma layer of C. moschata. The hull-less F2 progeny of Zhou Waltham
Butternut shows a residual lignification comparable to Type 2 in C. pepo (Pachner
and Lelley unpublished results).
Fig. 16.5 Hulled seed coat (a) with all three testa layers, i.e. hypodermis (2), sclerenchyma (3)
and aerenchyma (4) strongly lignified; Type 2 hull-less seed coat (b) with a continuously
lignified single sclerenchyma cell-layer; Type 3 seed coat (c) with discontinuous sclerenchyma
cell-layer; Type 4 hull-less seed coat (d) with all cell layers collapsed
In his study, Zraidi (2005) followed the segregation of only the non-
hulled types in three different F2 populations. In the F3, segregation pat-
terns of the selfed generations of Type 2 and Type 3 seed phenotypes did
not give a clue about the genetic determination of residual lignification.
They produced all three hull-less seed types in irregular ratios. Residual
lignification was found even in the selfed progeny of completely hull-less
genotypes. One major reason for a possible misclassification of F2 seeds
could be the substantial within fruit variation of residual lignification
(Fig. 16.6). Within-fruit variation of seed type was reported by Grebenšči-
kov (1950) and Teppner (2000), and within fruit variation could be observed
even in Type 1 segregants in Zraidis material (Fig. 16.7, Pachner and Lelley
unpublished data). The difficulty in classifying hull-less seeds notwithstand-
ing, the existence of modifying genes is also demonstrated by the prevalence
of numerous breeding lines in the US that show a preponderance of seeds
representing one or the other of the three hull-less seed types, Type 2, Type
3, or Type 4 (Loy, unpublished observations).
In histological preparations of the progenies of some of the hulled (Type 1)
segregants Zraidi (2005) found a second lignified sclerenchyma cell layer
(Fig. 16.8a) assembled on the top of the primary one, which is typical for the
seeds of the hulled parental genotypes (Fig. 16.5a). This second sclerenchyma
Fig. 16.6 Range of variation

of residual lignification of
Type 3 hull-less seeds in a
single fruit
Fig. 16.7 Within-fruit

variation of seed
lignification in Type 1
segregants
cell layer exhibited variation frequently observed in the testa of Type 3 hull-
less segregants (Fig. 16.8b,c). He did not exclude the possibility, that the same
N allele is accountable for the existence of this second sclerenchyma cell layer
in the hulled as for the single continuous sclerenchyma layer in Type 2 hull-less
segregants (Fig. 16.5b).
Fig. 16.8 Seed coat histology of hulled segregants (Type 1) of a cross hulled hull-less
pumpkin (Zraidi 2005) with two sclerenchyma cell layers (a) and varying expression of the
second lignified sclerenchyma layer (b and c)
Zraidis observations lead to the conclusion that Schöniger’s (1950) assump-

tion of a second incompletely dominant gene (N) might indeed be mainly
responsible for the residual lignification of genotypes homozygous recessive
(hh) for the major lignification gene. Nevertheless, seasonal differences in the
expression of the hull-less phenotype attributed to differential gene expression
by Stuart (1983) and within-fruit variation, which may become visible in
segregating progenies in all four seed type categories, so far has not allowed
unequivocal clarification of the genetic determination of residual lignification.
16.4 Current Goals of Oil Seed Pumpkin Breeding

Although the major use of hull-less seeded pumpkins has been for oil
production in central Europe, there is an expanding market for the seed as
food, especially as snack food and in confectionary products. In addition, it is
conceivable that a sandwich spread could be developed and marketed much in

the way peanut butter is utilized in North America (Curtis 1948). Although
much of the acreage of oil seed pumpkin in Europe and China is associated with
small farms and hand cleaning of seeds, for products of pumpkin seeds to
continue to be competitive with foods from other crops such as peanut, soybean
and sunflower, seed yields must be elevated and varieties have to be bred that
are resistant or tolerant to some of the more common diseases.
16.4.1 Increasing Seed Yield
Currently, the most popular varieties of oil seed pumpkins being cultivated in
Europe are rather large in size weighing 3–7 kg (Winkler 2000). Smaller-fruited
varieties have been introduced (Berenji 2000; Winkler 2000), but varieties with
larger fruit are apparently preferred, presumably because many farmers still
remove seed from the fruit by hand. Large fruits tend to have larger seed and a
more open cavity with looser placental tissue, so that it is easier to remove the
seed by hand. In pumpkin, changes in total plant biomass (biological yield)
produced per hectare is largely a function of cultural conditions such as fertility,
weed control, climate, length of the growing season and plant density, rather
than a function of genetic variation for greater photosynthetic efficiency. Thus,
for genetic improvement in seed yield, strategies must be employed to increase
the proportion of photosynthate partitioned into reproductive versus vegetative
tissues, and in particular, assimilates being partitioned into seed. Most
large-fruited oil seed pumpkin varieties are inefficient in this context, mostly
because the ratio of pericarp tissue to seeds is extremely high.
16.4.2 Components of Yield
The two overriding factors contributing to crop yield are production of total
plant biomass (biological yield) and the harvest index (HI) or proportion of
biological yield that is converted into reproductive biomass. Because changes in
biological yield are largely brought about by changes in cultural techniques,
genetic increases in yield among the major crop species have largely been
accomplished by increasing the harvest index (Donald and Hamblin 1976).
High HI values for fruit load in pumpkin can be achieved by breeding more
compact (bush) cultivars that produce a heavy fruit load on a restricted vine
(Broderick and Loy 1990). In the case of seed production, not only is prolific
fruit production per unit area important (high HI), but also the proportion of
total assimilate in the fruit that is partitioned into the seed. Two indices have
been developed to measure the efficiency of seed production within the fruit
(Loy 1991), the ‘seed index’ (SI), defined as seed dry biomass divided by total
fruit biomass, and seed dry biomass per kg fruit fresh weight (FW), a seed yield
efficiency measure that Nerson (2002) termed the ‘seed yield index’ (SYI). To
calculate these indices it is necessary to determine fruit fresh weight, % dry
weight (DW) of mesocarp tissue, and seed FW and DW per fruit. In addition, it
is usually important to select for seed size, and so seed size (mg) and in some
cases, individual parameters of seed size (seed width, length and thickness) are
also important variables to consider. In breeding results obtained at the Uni-
versity of New Hampshire from 1995 to 1999, seed yield per fruit was positively
correlated with fruit size (r = 0.68), but seed yield per fruit did not continue to
increase in fruit larger than about 3 kg (Fig. 16.9A). In a similar fashion, seed
size was positively correlated with fruit size (r = 0.58), but seed size did not
progressively increase in the best selections in fruit larger than about 2.5 kg
(Fig. 16.9B). On the other hand, the highest seed yield indices (SYI) were
obtained in fruit weighing between 0.5 and 1.5 kg (Fig. 16.9C). Both the seed
index and seed yield index are highly correlated with seed yield (Chretien and
Loy 2000), but the seed yield index is an easier parameter to measure and gives
as high or higher correlations to seed yield than the seed index. In breeding work
at the University of New Hampshire, seed yield indices of small-fruited
(0.7–1.5 kg) lines have been increased from 17 to 20 g seed/kg in initial breeding
lines (Loy 1991) to 50–65 in more advanced lines developed from additional
breeding cycles (Chretien and Loy 2000; Cui and Loy 2002). Winkler (2000) has
recently reported increasing seed yield indices from 15 in older cultigens to 30 in
newer breeding lines of oil seed pumpkin.
Fig. 16.9 Relationship of fruit fresh weight to seed DW per fruit (a), seed size (b), and seed
yield index (c) in seed pumpkin selections; seed yield index = g seed/kg fruit FW
16.4.3 Seed Size and Seed Number
Large seed size should not be a necessity for oil seed pumpkins harvested
mechanically as long as a high proportion of seeds per fruit is recovered during
harvesting, whereas larger seeds in the range of 170–220 mg are more desirable
for the snack seed industry. High seed yield indices and therefore high seed
yields are associated with fruit weights in the 0.5–1.5 kg range, so this raised the
question of whether consistently large seed size could be achieved in smaller
fruit. In a study reported by Carle and Loy (1994), a large-seeded (size range of
202–259 mg) hull-less accession (PI 285611) from Poland with a mean FW of
5.2 kg was crossed to a small-fruited (0.6 kg) breeding line (NH29-13-5-4) with
relatively small seed (116–167 mg). In the F2 population generated from this
cross, seed weight was positively correlated with fruit weight, but the correla-
tion was not strong (r = 0.48). Fruit weight was also positively correlated with
seed width (r = 0.40) and seed length (r = 0.54), but showed little association
with seed thickness (r = 0.14). The correlations might have been higher with
more small-fruited segregants in the population, because only one fruit in the
entire population of 450 plants even approached the small size of NH29-13-5-4.
The results, nonetheless, suggested that relatively large seed size could be
attained in fruit much smaller than PI 285611, especially if selecting for thick
seeds. One selection (NH396) from the F2 population above had a fruit size of
1.4 kg and relatively large seed size. Inbred lines developed from NH396 have
mean seed sizes in the 180–220 mg range and fruit size between 0.7 and 1.5 kg,
and have been used to produce productive F1 hybrid varieties (Cui and Loy
2002). Using thick or plump seeds as a major selection criterion has been further
fortuitous because poor tip fill is less of a problem in such seeds. For the snack
seed industry good tip fill is an important attribute because seeds with poor tip
fill do not puff well during the roasting process.
There are physical restrictions to how many moderately sized seeds can be
contained in a fruit with a fresh weight of 0.7–1.5 kg. Maximum FW seed size as
determined by maximum seed coat expansion occurs about 20 days after
pollination (DAP, Vining 1999). Likewise, maximum fruit FW in a small-
fruited cultigen is attained by 20 DAP (Berg 2004). Maximum seed numbers
per fruit are determined by the number of ovules in the ovary, but because of
seed abortion, developed seeds per fruit are usually considerably less than the
maximum number of ovules. Maximum seed numbers vary according to geno-
type, but seed counts in individual fruit as high as 691 have been reported in
small-fruited cultigens (Carle and Loy 1996). In high yielding hull-less cultigens
having fruit weights between 1.0 and 1.5 kg and seed weights of 170–200 mg, the
seed cavity is almost completely filled and average seed numbers under field
conditions typically vary from about 275 to 425 (Cui and Loy 2002). It may be
possible to make small genetic gains in seed numbers in large-fruited cultigens,
and thereby increase seed yields. In the early 1950s, Neumann (1952) and
Mudra and Neumann (1952) reported increasing seed yields by 10% or greater
by selecting genotypes with four or five carpels, rather than the common three.
In general though, in examining seed number distribution in numerous hull-less
cultigens with a range of fruit size and moderately large to large seed, it has been
observed that high seed numbers per fruit are not strongly associated with fruit
size (Loy, unpublished data).
16.4.4 Bush Growth Habit
Genetically dwarfed pumpkins, referred to as bush, are characterized by shorter

internodes, thicker stems, longer and thicker petioles, earlier flowering, and a
higher ratio of pistillate to staminate flowers than typical vine genotypes
(Loy 2004). The potential advantages of the bush habit of growth are faster
leaf canopy development at high density planting because of their uniform
growth pattern, more uniform fruit maturation because only one or two fruits
are set at high density planting, higher harvest indices (fruit to total biomass),
and easier cultivation for weed control. At least some of these attributes were
recognized by early breeders of oil seed pumpkin. As mentioned before, in the
1930s Tschermak-Seysenegg incorporated the bush or short internode habit of
growth into oil seed pumpkin by crossing an oil seed pumpkin with a bush
marrow squash resulting in ‘Tschermak’s Ölkürbis’ (Teppner 2000). Another
European bush variety of note, ‘Giessener’, produces elongate fruit weighing
about 3.0–3.5 kg on a bush plant. Fruits contain an abundance of moderately
large seeds, but the seed yield index varies from low (14; Berenji 1986) to
moderately high (34; Loy, unpublished data). To increase uniformity in matura-
tion and greater productivity, two small-fruited bush varieties, ‘Sepp’ and
‘Markant’, were developed and released in Austria in the mid-1990s. Also in
the 1990s, the bush variety ‘Olinka’ was developed in former Yugoslavia
(Berenji 2000) and has also been registered in Hungary and Slovenia.
In a breeding program at the University of New Hampshire, USA, to
develop hull-less seeded pumpkins for the snack food trade, the hull-less seeded
variety ‘Tricky Jack’ was used as a source of the bush gene. In 1986 two small-
fruited, bush breeding lines of hull-less pumpkins developed from this program,
NH14-40-6 and NH55-7-20, were evaluated for components of seed yield using
a gradient density planting scheme with densities from 7,200 to 36,000 plants/ha
(Loy 1991). Fruit size was moderately decreased at plant densities above 12,000
plants/ha, with mean fruit size being reduced from 1.2 kg to 0.8–0.9 kg at the
highest plant density. On the other hand, fruit number per plant for the two
breeding lines was reduced by more than 50%, from 3.5 to 3.6 at the lowest
plant density to 1.5–1.7 at the highest plant density. Seed yields were highest,
768 kg/ha and 942 kg/ha, respectively, for NH14-40-6 and NH55-7-20 at the
highest plant density, but seed yield indices were very low (17.5–18.2). Planting
densities between 18,000 and 24,000 plants/ha have proven to be near optimum
for producing high yields in more productive varieties developed during the
1990s from this breeding program (Cui 2005; Cui and Loy 2002).
The greatest utility of the bush habit of growth has been for hybrid seed
production. Ethephon, a growth regulator, releases ethylene upon decomposi-
tion, converting monoecious plants to female flowering (Lower and Miller
1969; Robinson et al. 1970). Bush plants can be treated with ethephon early
in development, causing plants to produce only female flowers for an extended
period. Therefore, a bush line treated with ethephon and serving as a female
parent, can be planted adjacent to a male pollinator line to produce hybrid seed
without hand pollination. Vine plants cannot be efficiently converted to fema-
leness by this method. However, by using a vine breeding line as the male
parent, F1 hybrids are produced that have a phenotype intermediate between
the bush and vine growth habit. In Cucurbita pepo, bush-vine hybrids generally
display a bush habit early and a more vining growth habit later in the season.
For pumpkins, this growth habit is generally considered more favorable than
the homozygous bush growth habit. Although homozygous bush cultivars lend
themselves to high density planting and easier weed control, one disadvantage is
that bush cultivars often lack sufficient vegetative growth to support their fruit
load, and this can adversely impact mesocarp dry matter and seed yields
(Loy 2004). Peak mesocarp dry matter occurs about 30–35 days after pollina-
tion (Culpepper and Moon 1945), but near maximum seed fill requires about 55
days after fruit set (Vining and Loy 1998). In bush cultivars, peak vegetative
growth occurs shortly after flowering, and this is soon followed by progressive
leaf senescence (Broderick and Loy 1990; Cui 2005). If sufficient vegetative
growth and photosynthetic leaf area are not attained by the time of fruiting,
then photosynthates may be potentially limiting for later stages of seed fill.
However, if enough assimilates are translocated from leaves and stored as
starch in mesocarp tissue by 35 DAP, then remobilization of mesocarp reserves
to the developing seed may be sufficient to attain good seed fill (Loy 2000).
To justify hybrid seed production, hybrid varieties must perform appreciably
better than OP varieties in terms of uniformity, seed size and overall seed yields.
Berenji (1986) produced six inbred breeding lines by selfing six existing open
pollinated varieties, one of which was bush ‘Giessener’. All of the breeding lines
but one had fruit size greater than 2.0 kg. The inbreds were intercrossed to
produce 15 F1 hybrids which were evaluated for seed size, fruit weight, seed
weight per fruit, yield per plant, and percentage of oil. High parent heterosis
was obtained in 13 out of 15 hybrids for seed weight per fruit and fruit weight.
High parent heterosis was also evident in 7 out of 15 hybrids for seed yield
indices; however, the highest seed yield index was only 28.4, and all but three of
the hybrids had seed yield indices below 22 g/kg fruit FW. In spite of the low
seed yield indices, all hybrids showed high parent heterosis for seed yield per
plant, and in one instance, the seed yield was more than doubled in the hybrid.
Correlation analysis indicated that specific combining ability was more
important than general combining ability in affecting yield components.
Cui and Loy (2002) reported on a study of heterosis in two experimental

hybrids (NH1050 and NH1051), using parents that had been selected for high
seed yield indices. Fruit biomass per plot was 32% higher in hybrid NH1051
than in the highest yielding parent. Mean fruit FWs of 1.49 kg for NH1050 and
1.33 kg for NH1051 were 28 and 32% higher, respectively, than in the largest
fruited inbred parents. Seed yields per plot in both hybrids were about 25%
higher than the highest yielding parent, but yield differences were statistically
significant only for NH1051, with plot yields extrapolating to 2,088 kg/ha.
Mean seed numbers per fruit were 428 for NH1050 and 396 for NH1051, and
both hybrids exhibited highly significant high parent heterosis for seed number
(24 and 35% increases) but not seed size, agreeing with previous observations by
Carle and Loy (1996). Coefficients of variation for mean seed size among fruit
were lower in NH1051 (3.7%) than in either parent (6.6 and 8.2%), reflecting
greater seed uniformity of the hybrid. Although no high parent heterosis was
exhibited for seed yield indices, the seed yield indices were high for all cultigens,
ranging from a low of 43.4 to a high of 60.7 g seed/kg fruit FW. Two bush vine
F1 hybrid seed pumpkins have been released by University of New Hampshire
for introduction into the commercial market, ‘Snackjack’ and ‘Snackface’. The
former variety produces small (0.8–1.4 kg) fruit and was developed as an
attractive ornamental pumpkin as well as for hull-less seed production. The
latter variety, a sister hybrid to NH1051, was developed for commercial seed
production for the snack food industry. It produces fruit in the 1.0–1.5 kg range,
seed size between 170 and 210 mg, and yields between 1,500 and 2,300 kg/ha (Cui
2005;) at a spacing of 18,000 plants/ha (0.3 1.8 m). Seed color in ‘Snackface’,
produced by the inner seed coat, is medium rather than the dark green preferred
for oil seed pumpkins.
16.4.5 Disease Resistance
Probably the two most prevalent diseases in regions growing C. pepo pumpkins
are powdery mildew (PM) and several viruses. Cucumber mosaic virus (CMV),
squash mosaic virus (SqMV), papaya ringspot virus (PRSV), zucchini yellow
mosaic virus (ZYMV) and watermelon mosaic virus (WMV) are usually the
most prevalent viruses in pumpkin. In many temperate regions PM does not
overwinter, and hence, infestations usually occur after fruit set. Because seed fill
is not completed until late in fruit development, heavy infestations of powdery
mildew can adversely impact photosynthate production and seed fill.
16.4.5.1 Resistance to Fruit Rots

Especially for harvesting hull-less seeded pumpkins by machine, fruit rots can
be the bane of successful production of hull-less seeds. Seed from a single rotten
fruit can produce off flavors in a seed batch, rendering seed inedible. Three
types of fruit rots are most prevalent in north temperate regions of pumpkin
culture, fusarium rots (Fusarium solanum and related species), bacterial leaf
spot rot (Xanthomonium campestris (Pammel) Dowson pv. cucurbitae (Bryan)
Dye), and black rot (Didymella bryoniae (Auersw.) Rehm). Although the fusar-
ium fungus has a wide host range, including corn and many legumes, it does not
appear to be a serious problem for hull-less genotypes (Loy, unpublished
observations). However, hull-less seeded pumpkins in particular appear to be
highly susceptibility to bacterial leaf spot. This disease is very subtle to detect
because leaf symptoms, appearing as small necrotic spots with a yellow halo, are
often fairly obscure. The lesions may coalesce and look similar to angular leaf
spot (Pseudomonas syringae pv. lachrymans) (Zitter et al. 1996). Initial symp-
toms on fruit appear as small, raised tan dots, but can expand into slightly
sunken tan spots, surrounded by a dark ring. Most of the lesions fail to
penetrate the fruit, but eventually penetration into the seed cavity will occur
on most fruit showing extensive symptoms. Tolerance of fruit to this disease has
been observed among ornamental pumpkins (Loy, unpublished observations),
and so attempts are being made to transfer this tolerance into hull-less seeded
breeding lines.
Most breeding lines developed at the University of New Hampshire do not
appear to be very susceptible to black rot. Recently, however, this disease has
become a major concern of oil-pumpkin growers in Austria (Huss et al. 2007).
Winkler (personal communication) has noted genetic variation for susceptibil-
ity for black rot among cultigens, so it appears that reasonable tolerance to this
disease can be introgressed into hull-less seeded pumpkins from existing
cultivars.
16.4.6 Expanding the Genetic Base in Oil Seed Pumpkins
Hull-less seeded pumpkin cultivars do not have natural resistance to the most
serious disease pathogens; thus, it is necessary to expand the genetic base of oil
seed pumpkins. According to Duchesne (1786) and Naudin (1856) C. pepo is the
most polymorphic species in the plant kingdom. Its fruits occur ‘in myriad of
shapes, sizes, and colors’ (Paris 1988). Genetic variation of Styrian oil-pumpkin
is, however, restricted due to selection for its specific seed-type and because of
its limited environmental range of cultivation. Because of its relatively simple
inheritance, the hull-less trait can be introduced into any cultivar type of C. pepo
(Paris 1986). The same might be true for the hull-less genotype found in
C. moschata (Zhou 1987). In many instances, however, it may prove desirable
to transfer useful genetic traits between species, especially those traits confer-
ring disease resistance. Successful interspecific crosses between C. pepo and C.
moschata are difficult but possible, especially with C. pepo as the maternal
parent (Castetter 1930; Erwin and Haber 1929; Robinson and Decker-Walters
1997; Pachner and Lelley unpublished results). Powdery mildew tolerance was
transferred from the wild species, C. okeechobeensis to C. pepo by using C.

okeechobeensis C. moschata as a genetic bridge (Robinson and Decker-
Walters 1997). This intermediate resistance to powdery mildew appears to be
due primarily to one incompletely dominant gene and has been introduced into
several squash and pumpkin cultivars. Loy (unpublished results) has intro-
duced this source of PMR into several hull-less seeded pumpkin breeding
lines that have been selfed to the F4 and F5 generations.
The first genes conferring tolerance in C. pepo to zucchini yellow mosaic
virus (ZYMV) originated from the C. moschata genotype ‘Nigerian Local’
(Provvidenti 1997). Further resistance against ZYMV was introduced into
C. pepo from the Portuguese landrace ‘Menina’ (Paris and Cohen 2000).
Studying the inheritance of resistance to ZYMV in five different C. moschata
genotypes of widely different geographic origin revealed at least five separate
loci that confer high tolerance against the virus (Pachner and Lelley 2004). Via
interspecific crosses these resistances are now available in C. pepo for further
breeding (Pachner and Lelley unpublished results). The C. moschata cultivar
‘Soler’ from Puerto Rico, in addition to having resistance against ZYMV, has
also provided resistance against powdery mildew to C. pepo (Pachner and
Lelley unpublished results). The Chinese hull-less genotype described by
Zhou (1987) also conferred resistance against cucumber mosaic virus (CMV).
Thus, C. moschata seems to be one of the most important genetic resources for
breeding of C. pepo.
While direct crossing of C. moschata with oil pumpkin has not been reported,
successful crossing of C. moschata with zucchini has been repeatedly achieved
(Robinson and Decker-Walters 1997). Crossing such an F1 with oil pumpkin
led to the introduction of several traits from C. moschata genotypes to oil
pumpkin (Pachner and Lelley unpublished results).
Recently the first consensus genetic map of C. pepo has been published (Zraidi
et al. 2007). This map has been constructed using mainly RAPD and AFLP
markers. Meanwhile, efforts to obtain SSR markers for Cucurbita, led to the
development of 500 such markers (Gong et al. 2008). These markers have been
used for updating the map of C. pepo as well as to develop a map for C. moschata.
The high transferability of the markers between C. pepo and C. moschata (around
90%) and also to C. equadorensis (up to 70%) suggest that these markers will be
very useful for a number of applications in the genus Cucurbita. Transferability to
other genera of the Cucurbitaceae family is under investigation. Altogether 5 SSR
markers have been found closely linked to the hull-less trait which could lead
eventually to the cloning of the gene responsible for testa lignification. SSR
markers can give a major boost for marker-assisted breeding in Cucurbita. One
SSR marker closely linked to resistance to ZYMV is now routinely used in

selection work (Gong et al. 2008).
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Chapter 17
Maize for Oil
Elizabeth A. Lee
17.1 Introduction
Maize is not traditionally viewed as an oilseed crop; actually maize oil is

considered a ‘minor’ oil among the traditional vegetable oils, representing
only about 3.4% of the 1998–1999 US market (Orthoefer et al. 2003). Maize
oil is a co-product of the wet and dry milling industries, which essentially
breaks down the maize kernel into starch, oil, protein, and fiber (for review see
Johnson and May 2003; Duensing et al. 2003). The US is the largest worldwide
producer of maize oil; Brazil, China, Romania, countries of the former Soviet
Union, former Yugoslavia, and South Africa also produce maize oil (Orthoe-
fer et al. 2003).
17.2 Maize Kernel Structure and Composition
The maize kernel is a mixture of maternal tissues (e.g., pericarp) and zygotic
tissues (e.g., embryo, aleurone, and endosperm) (Fig. 17.1). The embryo is
diploid (2n) while the aleurone (i.e., first cell layer of endosperm tissue) and
endosperm are triploid (3n). The triploid nature of the aleurone and endosperm
result from two identical maternal gametes (i.e., polar nuclei, the result of
megasporogenesis) fusing with one paternal gamete (i.e., sperm nuclei, the
result of microsporogenesis).
The typical maize kernel, on a dry weight basis, is composed of 61–78%
starch, 6–12% protein, 3.1–5.7% oil, 1.0–3.0% sugar, and 1.1–3.9% ash
(Miller 1958; Watson 2003). Most of the starch is associated with the
endosperm (<95%), while the embryo contains high levels of protein
(26%), oil (83%), sugar (70%), and ash (80%) (Earle and Curtis
1946; Watson 2003). The typical fatty acid profile of a maize kernel is
57.9% linoleic acid, <1% linolenic acid, 25.2% oleic acid, 11.6% palmitic
E.A. Lee (*)

Department of Plant Agriculture, University of Guelph, Guelph, Canada
e-mail: lizlee@uoguelph.ca

494 E.A. Lee
Fig. 17.1 Diagram of a

maize kernel. The pericarp Pericarp
is maternal tissue, the
endosperm and aleurone are
triploid tissues, and the Endosperm
embryo is diploid
Aleurone
Embryo
acid, and 1.8% stearic acid (Dunlap et al. 1995; White and Weber 2003).
Most of the genetic research on maize kernel oil has focused on total
content rather than composition, the inheritance of which is highly quanti-
tative; however, several single gene mutations influencing fatty acid profile
have been identified. Linoleic acid content is controlled by a single recessive
mutation, linoleic acid1 (ln1) (de la Roche et al. 1971). High oleic acid
content (57% vs. 27%) is due to the recessive mutation oleic acid content1
(olc1) (Wright 1995).
17.3 Modern Maize Breeding
Because of the hybrid nature of the crop modern temperate maize breeding in
the US and Canada has evolved into two very distinct activities: inbred line
development and hybrid commercialization (Duvick and Cassman 1999; Lee
and Tollenaar 2007; Fig. 17.2). Maize breeding in North America, unlike
breeding activities for many other crop species, has been done primarily in the
private sector for the past 30+ years. Movement of this activity out of the
public domain has been accompanied by legal protection of the commercial
germplasm through legally binding ‘use agreements’, US patents, and the US
Plant Variety Protection Act (PVP act).
17.3.1 Germplasm
The genetic base of North American hybrid maize industry represents only a
small portion of the entire Zea mays gene pool. There are 250–300 races of
maize (Brown and Goodman 1977), of which only one, the Corn Belt Dent
(CBD), is the predominant source of commercial germplasm (Goodman 1985).
17 Maize for Oil 495
Maize Breeding 3rd party

proprietary
Parental inbred lines inbred lines
of successful
hybrids
Inbred Line Hybrid

Development Commercialization
New hybrids
New inbreds
Sold to farmers
Fig. 17.2 Overview of a modern maize breeding program. (from: Lee and Tollenaar 2007)
Of the hundreds of open-pollinated varieties (OPVs) of CBD that were grown

up to the 1940s, only half a dozen or so can be considered as significant
contributors to current inbred lines. But the overwhelming majority of the
inbred lines trace their pedigree back to only two OPVs, Reid Yellow Dent
and to a far lesser extent Lancaster Surecrop (Goodman and Holley 1988; Tracy
and Chandler 2006; Lee and Tracy 2009)
One of the consequences of the hybrid concept was the development of
heterotic patterns, also referred to interchangeably as heterotic groups. These
groups were ‘created’ by breeders as a means of maximizing the amount of
hybrid vigor and ultimately grain yield in a more predictable manner (cf., Tracy
and Chandler 2006). Modern hybrids then are the result of crossing an inbred
line from one heterotic pattern with an inbred line from a different heterotic
pattern. Today, inbred lines are classified into heterotic groups and are further
sub-divided into families within a heterotic group. Classification of heterotic
patterns is generally based upon several criteria such as pedigree, molecular
marker-based associations, and performance in hybrid combinations (Smith
et al. 1990), and has resulted in somewhere between two and seven distinct
heterotic patterns being described (Smith and Smith 1989; Troyer 1999; Lu and
Bernardo 2001; Gethi et al. 2002; Mikel and Dudley 2006). We have chosen to
represent the germplasm as three main heterotic patterns ‘Stiff Stalk’,
‘Lancaster’ and ‘Iodent’, and a miscellaneous category of lines that do not fit
into the three primary heterotic patterns in the northern and central US and
Canadian corn growing regions. Most conventional inbred line development
has focused on ‘recycling’ (i.e., intermating) lines within a heterotic pattern and
even to within a heterotic family; however at least two novel heterotic patterns
496 E.A. Lee
have arisen: ‘Maiz Amargo’ and ‘Commercial Hybrid’ derived germplasm

(Mikel and Dudley 2006; Lee and Tracy 2009).
17.3.2 Heterosis and the Inbred/Hybrid Concept
Hybrid maize traces its roots back to experiments on heterosis and inbreeding
conducted by Shull (1908, 1909) at Cold Spring Harbor Laboratories in New
York and East (1909) at Connecticut State College. They observed that when
maize plants were self-pollinated (i.e., inbred) in successive generations, their
vigor and grain yield rapidly deteriorated (Shull 1908; East 1909). However,
when two inbred lines from unrelated populations were crossed, both vigor and
grain yield of the F1 hybrid often exceeded that observed for the original source
populations (Shull 1908). It was these observations, made nearly 100 years ago,
and methodology outlined by Shull (1909) that gave rise to the modern hybrid
maize industry (cf., Crow 1998).
Since Shull’s initial papers there has been debate about the underlying
genetic mechanism of heterosis. The two main theories are the dominance
theory and the overdominance theory. The dominance hypothesis attri-
butes heterosis to the accumulation of favorable dominant genes or mask-
ing of deleterious recessive alleles in the hybrid (Davenport 1908; Bruce
1910; Keeble and Pellew 1910). In quantitative genetic terms, heterosis
results when there is some degree of directional dominance (d) and the
parents differ in gene frequency (Bruce 1910; Falconer 1981). The dom-
inance hypothesis can be expressed in terms of a single-locus (B) with no
epistasis as
Heterosis ¼ d fa þ ðaÞg=2 (17:1)
where a and –a are the genotypic values of the parental genotypes (B1B1 and
B2B2) and d is the genotypic value of the non-parental genotype (B1B2). The
dominance theory is consistent with recent genomic evidence of differences in
genic content between maize inbred lines (Fu and Dooner 2002; Song and
Messing 2003; Brunner et al. 2005), and has been demonstrated as the under-
lying mechanism of a heterotic response for grain yield in a quantitative trait
locus mapping study (Graham et al. 1997). The other hypothesis,
over-dominance, argues that the heterozygous combination of the alleles at a
single locus is superior to either of the homozygous combinations (Shull 1908;
East 1908). The over-dominance hypothesis, unlike the dominance hypothesis,
does not require the presence of either linkage or the involvement of multiple
loci for heterosis to be expressed, nor is it necessarily based on classic Mendelian
genetics. However, like the dominance hypothesis, it also requires that the
parents differ in gene frequency. While there is no direct evidence in support
of this hypothesis in the literature, it has not been completely rejected as an

underlying genetic cause.
17.3.3 Inbred Line Development

Most if not all inbred line development activities in North America involve the
use of the pedigree method of breeding (Duvick et al. 2004; Mikel and Dudley
2006; Lee and Tollenaar 2007; Fig. 17.3). Pedigree breeding as it is structured in
the commercial sector is akin to reciprocal recurrent selection (RRS) (Hallauer
and Miranda 1988; Duvick et al. 2004). Breeding crosses tend to be made by
crossing inbred lines within a heterotic pattern. Inbred lines from the other
heterotic patterns are used to improve the heterotic pattern represented by the
breeding cross. This type of breeding scheme (i.e., akin to RRS) allows maize
breeders to improve both additive and non-additive genetic effects, resulting in
greater overall genetic gains (Comstock et al. 1949).
Stiff Stalk
Parental inbred lines
heterotic
of successful hybrids
pattern
“Release” new
inbred lines”
Inbreed and
eliminate obvious
2-parent cross
defects.
Evaluate in AxB
multiple locations
(limited no.).
× Lancaster
× Iodent S2 lines
S5 lines
× Lancaster
× Iodent
Select & continue

to inbreed.
Evaluate in multiple
locations (limited no.).
Fig. 17.3 Typical inbred line development scheme depicting a 2-parent breeding cross invol-
ving two inbred lines from the Stiff Stalk heterotic pattern. (from: Lee and Tollenaar 2007)
Marker assisted breeding, a variation of the pedigree method, is being used

by some companies. Based on the phenotype data from early generation testing,
molecular marker genotyping is used in conjunction with off-season nurseries
to reduce inbred development time (cf., Lee and Tracy 2009). In recent years
there has been a renewed interest in using doubled haploids in maize inbred line
498 E.A. Lee
development, reducing the time of the inbreeding process (cf., Lee and Tracy
2009).
Compared to other crops backcrossing has not been heavily used in maize
breeding. Backcrossing has been used to incorporate disease resistance genes
into elite inbred lines or to transfer inbred nuclear genotypes into male sterile
cytoplasms (Hallauer et al. 1988). However, with the widespread use of trans-
genes in maize breeding (e.g., herbicide resistance genes, insect resistance
genes), backcrossing coupled with molecular markers for rapid recurrent parent
genome recovery has become a standard tool of the maize breeder (Lee and
Tracy 2009). Most commercial maize breeding programs have adopted the
process of developing new inbred lines from non-transgenic germplasm, and
then as new inbreds show promise they will begin backcrossing in desired
transgenes, at the same time maintaining the original non-transgenic inbred.
Thus, if a particular transgene is removed from the marketplace, or the use
agreements are no longer valid the new inbred is not lost. This also permits the
sale and production of non-transgenic versions of the new hybrids in locations
that disallow the use of transgenes.
17.3.4 Hybrid Development

Commercial maize grain yields during the hybrid era (1939 onward) have
increased more than 6-fold (Lee and Tollenaar 2007). This increase is not
exclusively due to genetic improvement, as there have been substantial changes
to agronomic practices during this 65–70 year period (e.g., plant population
density and distribution, herbicides, fertilizers). As the agronomic practices
changed, breeders incorporated these changes into their testing programs,
meaning that realistically 100% of the increase in grain yield is actually due to
the interaction between genetics and agronomic practices (Tollenaar and Lee
2002).
In contrast to inbred line development, hybrid commercialization generally
involves a multi-tiered testing system (Duvick and Cassman 1999). More hybrid
combinations are tested in fewer environments during the early testing phase,
while in the later testing phases fewer hybrid combinations are tested in more
environments. Again, grain yield is the primary trait of interest, testing for grain
yield is always done in hybrid combinations, and testing is done using the most
current agronomic practices. The specific traits that are assessed are:
(1) machine harvestable grain yield adjusted to 15.5% grain moisture (bu ac1
or Mg ha1); (2) grain moisture at harvest (%); (3) test weight, a measure of
bulk density (lbs bu1 or kg hl1); (4) broken stalks, dropped ears, root lodging;
(5) days to 50% anthesis, days to 50% silking, and the interval between silking
and anthesis; (6) germination under cold, wet conditions (i.e., cold germ test);
(7) disease resistance (ear, stalk, and leaf diseases); (8) general plant appearance
(cf., Lee and Tracy 2009).
17.4 Recurrent Selection
Noticeably absent from the methods for inbred line development outlined
above is recurrent selection (RS). While RS has been widely used in the public
sector for germplasm improvement, it is not a method that is routinely utilized
by the commercial maize breeding sector for inbred line development, circa
2007. However, RS was the breeding method used in the Illinois High-Oil/Low-
Oil (IHO/ILO) long-term selection experiment and therefore warrants some
general discussion regarding methods and philosophy.
The general goal of RS programs is to increase the frequency of desirable
alleles while maintaining genetic variation in the population (Hallauer and
Miranda 1988). In maize breeding, a population is a heterogenous mixture of
heterozygous genotypes derived by intermating inbred lines and/or OPVs.
Populations are maintained by randomly intermating individuals in the popu-
lation or allowing open-pollination under isolation (i.e., at least 200 m from
other maize). Recurrent selection programs are cyclical with a cycle consisting
of a generation in which selection takes place followed by recombination of the
selected individuals or families. In academic research the term RS generally
indicates a closed population (i.e., no new germplasm being incorporated)
unless otherwise specified. Hallauer and Miranda (1988) and Bernardo (2002)
outline numerous RS programs.
Divergent recurrent selection is a form of RS in which starting from an initial
closed population a trait is selected in opposite directions and populations
divergent for that trait are developed. Divergent RS experiments can be useful
in understanding the limits of selection and also the relationship between the
selected trait and unselected traits that change in response to selection (cf., Lee
and Tracy 2009).
17.5 The Illinois High-Oil/Low-Oil Long-Term

Selection Experiment
The Illinois high-oil/low-oil (IHO/ILO) long-term selection experiment is per-

haps the best known example of a divergent RS experiment in which there have
been more than 100 generations of selection for kernel oil content (Dudley and
Lambert 2004). G.C. Hopkins, a chemist by training, initiated the experiment in
1896 to determine if it was possible to change the chemical composition of a
maize kernel (Hopkins 1899). Hopkins actually initiated two divergent selection
experiments, one for oil and one for protein. Both selection experiments were
started using an OPV called Burr’s White. The selection experiment has con-
tinued since, except for 4 years during the Second World War. Several compre-
hensive summaries of the IHO/ILO experiment have been published that
contain details regarding selection intensity, methods of chemical analysis,
and breeding procedures (for details see Dudley et al. 1974; Dudley and
500 E.A. Lee
Lambert 2004). Seven researchers have shepherded the IHO/ILO experiment

from cycle 0 through cycle 100: C.G. Hopkins, L.H. Smith, C.M. Woodworth,
E.R. Leng, D.E. Alexander, R.J. Lambert, and J.W. Dudley. What essentially
started out as a relatively simple experiment, to determine if it is possible to
change to chemical composition of a maize kernel through selection, resulted in
the development of a very unique set of germplasm for quantitative genetic
studies. Results from these studies and their implications will be briefly sum-
marized and discussed below.
Obviously the answer to C.G. Hopkins question was yes, that through
selection the chemical composition of a maize kernel can be altered (Fig.
17.4). The oil content of Burr’s White, as measured by Hopkins at the start of
the experiment was 4.69%. After over 100 cycles of selection for oil content in
the high direction, the mean oil content of the IHO population was 20.37%,
which translated into a gain per cycle of selection of 0.17% (Dudley and
Lambert 2004). Progress in the low direction, however, did not mirror that of
the high. For the first nine cycles of selection, progress in the low direction
proceeded at a rate similar to that of the high (0.22% cycle1), however
beyond cycle nine progress in the low direction ranged between 0.05 and
0.01% cycle1 with progress essentially stopping after cycle 76 with a mean
oil content of 0.35% (Dudley and Lambert 2004). Selection for low kernel oil
content had reached a selection limit and one, which most likely, was
Fig. 17.4 Illinois long-term high-oil/low-oil selection experiment 100+ generations. Plot of
mean oil concentration against generation (i.e., cycle) for Illinois High Oil (IHO), Reverse
High Oil (RHO), Switchback High Oil (SHO), Illinois Low Oil (ILO), and Reverse Low Oil
(RLO). (from: Dudley 2007)
anticipated, based on biology and foreshadowed by several earlier quantitative

genetic examinations of the selection experiment (Winter 1929; Dudley and
Lambert 1969).
After 48 cycles of ‘forward’ selection for high-oil and low-oil, E.R. Leng
initiated ‘reverse’ selection to determine how much residual genetic variation
remained in the populations available for selection to act upon (Leng 1962). The
reverse high-oil (RHO) population started with cycle 48 of ILO with selection
proceeding in the high direction and the reverse low-oil (RLO) population
started with cycle 48 of IHO with selection proceeding in the low direction
(Fig. 17.4). After seven generations of selection in RHO, a fifth selection
experiment was initiated called ‘switchback’ high-oil (SHO). Selection in
RHO was immediately effective in shifting the mean oil content downwards,
however, selection in the RLO had little effect in the first several generations,
with marked progress being evident only after the third generation of selection
(Leng 1962). The effect of selection in the SHO exhibited the same lag that was
exhibited in the RLO, only after four generations of selection in SHO did the
mean oil content shift upwards (Leng 1962). The two ‘reverse’ populations and
the ‘switchback’ population demonstrated that there was still sufficient residual
genetic variation upon which selection could act to drive the phenotype in the
opposite direction. What was perhaps the most surprising was that as much
genetic variability was present after long-term selection as existed in the original
unselected population (Leng 1962).
Progress from selection in IHO and ILO was due to the accumulation of a
large number of small favorable alleles in coupling phase linkage, that were
present in the original Burr’s White OPV at a low frequency (Dudley 2007).
Accompanying these changes in kernel oil content, were changes in starch and
protein composition of the kernels. During the first 70 cycles of selection in IHO
and ILO, protein concentration increased from 109 to approximately 150 g/kg
(Dudley et al. 1974). From cycle 65 to 100 of IHO oil content increased from 161
to 224 g/kg, protein concentration showed a significant increase from 148 to
158 g/kg, while starch content significantly declined from 435 to 336 g/kg
(Dudley and Lambert 2004). The IHO/ILO germplasm has been used in several
quantitative trait loci (QTL) studies, the results from which confirm the findings
of the classic quantitative genetic studies and the correlated responses
(cf., Dudley 2007).
17.6 Other Breeding Programs for High-Oil
The IHO/ILO long-term selection experiment was not the only breeding popu-
lation developed for kernel oil content. In the 1950s, D.E. Alexander initiated
several ‘new’ breeding programs for high-oil maize. There have also been
several forays into developing commercial high-oil hybrids through various
approaches.
502 E.A. Lee
17.6.1 ‘Other’ Recurrently Selected High-Oil Populations
D.E. Alexander initiated five ‘new’ breeding populations for high-oil selection
in the 1950s. The intent was not to duplicate the IHO selection experiment, but
rather to develop other sources of ‘high-oil genes’ for breeders to use. The other
compelling reason for creating these populations was to examine the negative
association between grain yield and oil content that was present in the IHO
population (note the decline in starch content in IHO mentioned above)
(Lambert et al. 2004).
(1) Alexho Synthetic served as the source for three additional selection
experiments: Alexho Single Kernel, Alexho Elite (AE), and Ultra High-
Oil (UHO).
(2) Disease Oil (DO) – developed to combine improved resistance/tolerance to
leaf blights and stalk rots with high-oil.
(3) Arnel’s Reid Yellow Dent (ARYD) – a selection of 363 ears from the OPV
Reid Yellow Dent that was grown on the Arnel farm in 1964.
(4) Stiff Stalk Synthetic High-Oil (RSSSCHO) – comprised of intermating
three improved versions of Stiff Stalk Synthetic.
(5) Iowa-2-Ear High-Oil (BS10HO) – a two-eared population developed by
analyzing 10,000 kernels from BS10(FR)C2.
Work with these populations showed similar responses to selection for oil
content as the IHO population. However, unlike the IHO population, two
kernel mutations (waxy and floury), which affect starch composition and
protein content, respectively, arose in the later cycles of Alexho Synthetic.
17.6.2 Commercial Breeding Activities for High-Oil
The first major attempt at developing commercial high-oil hybrids was in the
late 1940s by converting the widely grown double-cross hybrid, US13, into a
high-oil hybrid. This involved backcrossing IHO genes into the four parental
inbred lines: Wf9, 38-11, Hy2, and L317. Rather than measuring oil content
during the backcrossing process, embryo size was used as an indicator of oil
content. In the end, the converted US13 was 5% lower in grain yield, 8%
higher in protein content, and 30% higher in oil content (Jugenheimer 1961).
Starting in 1946, Funk Bros. Seed Co. began a high-oil breeding program that
continued until 1972. The initial breeding approaches involved both RS and
backcrossing the IHO genes into conventional inbred lines. The RS efforts were
mildly effective (58–63 g/kg oil content), while the backcrossing approach
produced inbred lines in the 70–100 g/kg oil content range. Funk Bros. used
these lines to produce three high-oil double cross hybrids. Unfortunately these
hybrids were 10% lower in grain yield than Funk Bros.’s widely grown hybrid
(Lambert 2001; Lambert et al. 2004).
17.6.3 TopCross1 Method for High-Oil Maize Hybrids
TopCross1 and TC Blend1 seed corn are proprietary technologies of DuPont

Quality Grains for producing high-oil maize while minimizing the yield dis-
advantages that traditionally accompany high-oil maize production (Bergquist
et al. 1998, 1999). The approach utilizes a blend of two genotypes in a unit (i.e.,
bag) of seed maize. One genotype, comprising 90–92% of the seed in the blend,
is a hybrid that is designated as the ‘grain parent’ and is male sterile, meaning
that it produces no pollen. The second genotype, representing 8–10% of the
seed in the blend, is a called a ‘pollinator’. Unlike the grain parent, this genotype
does produce pollen and is solely the source of pollen within the TopCross1
grain production field. The ‘pollinator’ is a high-oil genotype (generally a
synthetic population) in the 120–150 g/kg oil content range (Lambert 2001).
The resulting F1 kernels on the ‘grain parent’ will have higher oil content due to
the genetics of the ‘pollinator’, a phenomenon called xenia where the genotype
of the pollen parent affects the phenotype of the grain. Because of the xenia
effect it is important to isolate TopCross1 grain production fields from other
maize fields to minimize the amount of pollen contamination from non-high oil
maize. Reports suggest that in general maize kernel oil contents between 60 and
80 g/kg can be achieved with this technology (Lambert et al. 2004), but there
still appears to be a significant yield disadvantage (Thomison et al. 2002).
Epilogue
While maize is a ‘minor’ oil crop, the breeding efforts for high-oil maize have
made contributions beyond maize. The long-term selection experiment for
high- and low-oil content has permitted testing of fundamental quantitative
genetic principals that pertain to selection in both plants and animals. Both the
IHO/ILO experiment and other high-oil breeding efforts have contributed
breeding approaches and analytical technologies to other oilseed breeding
programs. The use of NMR technology for single kernel selection oil content
(Bauman et al. 1963) and backcross conversions are just two examples.
Dedication The author would like to dedicate this chapter to John W. Dudley and Robert J.
Lambert for their perseverance and commitment to seeing the Illinois High-Oil/Low-Oil long-
term selection experiment through 100 generations of selection.
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Chapter 18
New Crops Breeding: Lesquerella
David Dierig and Dennis T. Ray
18.1 Introduction
Lesquerella species contain a seed oil which is approximately 55% lesquerolic

acid, a 20-carbon long fatty acid with a single hydroxyl group and double bond,
and has a similar hydroxy fatty acid (HFA) profile as castor oil. Large markets
exist for hydroxylated oils as feedstocks for lithium greases, polymers in paints
and coatings, base stocks for lubricants, nylon-11, hydraulic fluids, and appli-
cations in the personal care industry (Roetheli et al. 1992). The hydroxyl group
of these oils makes them prime candidates as additives to diesel fuel to improve
lubricity (Naughton 1992). Goodrum and Geller (2005) demonstrated that
lesquerella oil has superior performance compared to castor, soybean, and
rapeseed methyl esters at concentrations as low as 0.25% in reducing wear
and damage to diesel engines, primarily with fuel injection systems. Castor oil
also contains high amounts of HFAs, but the main HFA, ricinoleic acid, is two
carbons shorter than lesquerolic acid, which imparts slightly different physico-
chemical properties to the oil. Lesquerella could be established as a reliable,
domestic oilseed supply of HFAs for a variety of industrial applications
(Roetheli et al. 1992) and at the same time provide an alternative crop for
farmers and increase local profits. Lesquerella will not replace current
commodity crops but instead will be placed in a rotation with these crops,
e.g., a 2-year, 3-crop rotation of lesquerella, grain sorghum, and cotton.
Lesquerella is a genus of the Brassicaceae family with species ranging primarily from
the arctic to southern Mexico, with another dozen species occurring in South
America. The greatest concentration of taxa are in the southwestern United States
and Mexico, where L. fendleri originates, and in the Rocky Mountain and
D. Dierig (*)
USDA-ARS-Arid Lands Agricultural Research Center, Maricopa, AZ, USA
e-mail: david.dierig@ars.usda.gov

508 D. Dierig and D.T. Ray
intermontane basin region of the western U.S. (Rollins and Shaw 1973;
Rollins 1993). L. fendleri (Fig. 18.1) contains the hydroxy fatty acid lesquerolic
acid (14-hyroxy-cis-11-eicosenoic acid, C20:1-OH) as the main component of its
seed oil. Other species with a second type of HFA in their seed oil, densipolic acid
(12-hydroxy-cis-9-cis-15-octadecadienoic acid, C18:2-OH) originate from the
eastern U.S. A third type of HFA called auricolic acid (14-hydroxy-cis-
11,cis- 17-eicosadienoic acid, C20:2-OH) is found primarily in one species from
Oklahoma.
Fig. 18.1 Flowers (left) of Lesquerella fendleri (Brassicaceae), and a lesquerella production
field (right) in Arizona
The genus Lesquerella was named in honor of paleobotanist Leo Lesquereux

(1806–1889), who came from Switzerland to the U.S. in 1848, and settled in
Columbus, Ohio. His specialty was the collection of fossil plants, especially in
connection with coal deposits. The taxonomy of Lesquerella, commonly called
bladderpod, was studied by Payson (1921) and later revised by Rollins (1955).
O’Kane et al. (1999), Al-Shehbaz and O’Kane (2002), and O’Kane and
Al-Shehbaz (2002) proposed that 75 species in the genus Lesquerella be trans-
ferred to the genus Physaria and all of the annual species of Lesquerella from the
southeastern U.S. be placed into a new genus, Paysonia. Their new synonym for
L. fendleri is Physaria fendleri (A. Gray). Public literature still refers to this plant
as lesquerella or L. fendleri and Rollins and Shaw (1973) noted difficulties in the
similarities between the two genera but also stated the problem of merging them
because of similarities with Lesquerella and another genus Alyssum. The
distinction for agronomic purposes and crop development is that L. fendleri is
a short-lived perennial that can be grown as an annual. Some other species of
Lesquerella and Physaria are perennial and do not flower until the second year
of growth. Physaria species are not as adaptable for domestication because of
this, even though they can be very productive seed yielders.
18 New Crops Breeding: Lesquerella 509
The first interest in Lesquerella species for domestication came in the late
1950s. USDA-ARS began a massive screening of over 200 families of oilseed
plants from the wild and described the new hydroxy fatty acids from this plant
(Jones and Wolf 1960; Smith et al. 1961). Plant exploration trips to collect
Lesquerella species were based on the fact that only occasionally does a plant
with an unusual oil composition also have high crop potential with no real
biological barriers for domestication. A series of articles The Search for New
Industrial Crops and The Search for New Industrial Oils were published in
Economic Botany and the Journal of the American Oil Chemists’ Society
between 1960 and 1984, and described lesquerella as well as other potential
new crops. The second article in The Search for New Industrial Crop series was
the first description of collections of Lesquerella made by USDA-ARS (Barclay
et al. 1962). A breeding program by Dr. D. Rubis at the University of Arizona
began in 1968 and continued until 1971, using the germplasm collected by
USDA. Dr. Rubis generously contributed his germplasm to the USDA, ARS
program in Arizona that began in 1986 by Dr. A.E. Thompson (Thompson and
Dierig 1994). The breeding program is still ongoing at the ARS facility in
Maricopa, Arizona.
L. fendleri is found in its native environment on calcareous soils in the
southwestern states of Arizona, New Mexico, and Texas, with a few collections
from southern Utah and Colorado by Rollins and Shaw (1973). Collections
from the states of Coahuila, Chihuahua, Nuevo Leon, Zacatecas, and
Durango, Mexico were made (Salywon et al. 2005; Rollins and Shaw 1973).
These populations were usually associated with moisture availability in mixed,
sparse vegetation, and were easily recognized by their glabrous siliques and
fused trichomes which set L. fendleri apart from other Lesquerella species.
There are 233 Lesquerella accessions available in the National Plant Germplasm
System (NPGS). One hundred and twenty of these are L. fendleri, and most of
these accessions were collected during the period from 1993 until 2002 through
trips supported by USDA-ARS (Dierig et al. 1996; Salywon et al. 2005). Prior to
this there were only 17 species represented and 21 accessions of L. fendleri in
NPGS (Thompson et al. 1992). In the USDA-ARS-ALARC working collection,
there are now 413 accessions of 57 Lesquerella and 17 Physaria species. One
hundred and thirty are L. fendleri accessions. A phenotypic evaluation of germ-
plasm available in NPGS was completed by Jenderek et al. (2008). The curator
within the NPGS for Lesquerella species is located at the USDA, ARS, National
Arid Land Plant Genetic Resources Unit – Parlier, California.
In Rollins’ review (Rollins 1993) of Lesquerella of North America, 83
species were included. Other species have since been discovered including
four by Rollins (1997), Rollins et al. (1995) and Anderson et al. (1997) as
well as others by O’Kane (1999) bringing the total number of North

American species to about 90.
Some Lesquerella species are on federal or state lists as rare, endangered, or
threatened species. One of these species, L. pallida, has been valuable to our
breeding program because of its high lesquerolic HFA content. This species is
also autofertile compared to the self incompatibility and open pollination of
L. fendleri. No collections have been made of this species since the original
collection in the 1800s, until a report by Nixon et al. (1983) on the rediscovery of
the species.
A number of Lesquerella and Physaria species have traits of interest for
genetic improvement, although none have the productivity of L. fendleri.
Apart from plant accessions our database contains over 10,000 records of
germplasm lines including various traits such as yellow seeds, non-shattering
selections, salt tolerance, male sterility, five petal plants, multi-locule silique
selections and other traits and crosses.
An important accomplishment has been to provide a large collection of genetic

diversity through plant germplasm collections. The numerous collection trips
throughout the U.S. and Mexico and subsequent evaluations provided valuable
diversity for the plant breeding program. Within these accessions, traits such as
flower color, autofertility, plant architecture, seed coat color, male sterility, and
seed oil characteristics have been identified. The inheritance of some of these traits
has been determined to allow their use in the breeding program (Dierig et al. 2001).
The improvement of oil content has made a significant contribution to the
commercialization process of lesquerella. Unimproved accessions of L. fendleri
have oil contents around 24%. The last germplasm line publicly released had
32% (Dierig et al. 2006a), and another line ready for release has 36% oil content.
The difference in oil yield between the unimproved and improved accessions is 86
liters/ha (56 gal per acre) oil versus 146 liters/ha (84 gal per acre) based on current
yields of 1800 kg/ha, respectively.
The oil profile of this species has been surveyed by Dierig et al. (2006b) and
lesquerolic HFA was highly variable. L. fendleri appears to have a limit of
around 67%. Hayes et al. (1995) reported that L. fendleri fills only 2 of the 3
triglyceride positions with HFA. The sn 2 position is left unfilled, which
explains the upper limit of two-thirds or 67% for HFA content. A few other
species of Lesquerella are able to fill all 3 positions and have HFA content of up
to 89%, which is near the level of castor. Current lines contain 60% lesquerolic
acid. A single gene mutation with high oleic and 0% lesquerolic acid was
recently discovered (Dierig et al. 2008, in prep.). This germplasm provides a
platform for examining a molecular approach for transforming lesquerella with
genes in the HFA biosynthetic pathway to increase the HFA content.
Interspecific hybrids between L. fendleri and two other species have been
developed, introgressing the high lesquerolic trait into L. fendleri. The hybrids
are not ready for public release until further improvements are made but have
over 75% lesquerolic acid (Dierig et al. 2004). Seeds per pod (silique) are fewer
than L. fendleri, but this is as expected since the other parent species had larger
but fewer seed, and the hybrid expressed mid parent values. The hybrids hold
promise for providing non-genetically modified traits not currently available in
L. fendleri such as high lesquerolic acid, autofertility, and an expanded
geographical area for commercial production.
18.5 Current Breeding Goals
Lesquerella fendleri plants are open-pollinated, highly genetically diverse,

and have no biological barriers to being a commercial crop. L. fendleri is
highly productive and it is felt that by exploring other areas of research the
crop has the potential to double in yields. A few traits that could be
further exploited will be discussed below, although this is not a compre-
hensive list.
18.5.1 Oil Content and Fatty Acid Profile
There have been two plant germplasm releases with improved total oil and
lesquerolic acid contents (Dierig et al. 1998, 2006a). Results from a recur-
rent selection program have consistently produced plants with oil content
between 40 and 45%. This appears to be the upper limit for the oil content
of this plant. However, this is the average value of seeds from a single
plant. There has yet to be a screening of single seeds for this trait; a
technique has just been developed, but will still be difficult because of
the small seed size (Isbell et al. 2008). A single seed weighs approximately
0.0006 g. Screening germplasm via single seeds for this trait should allow
faster progress in increasing oil content.
The upper limit for lesquerolic acid HFA content in L. fendleri appears
to be about 67% (described above). Some market applications may require
this to be kept at the limit if the material, such as nylon 11, requires a
difunctional triglyceride. Lines are being developed through the develop-
ment of interspecific hybrids with greater than 80% lesquerolic acid utiliz-
ing species with all three positions of the triglyceride (trifunctional) and
introgressing that trait into the hybrids. An improved line with these traits
would fit most of the same markets as imported castor. The other desir-
able goals would be to lower the unsaturated fatty acids linoleic and
linolenic acids.
18.5.2 Seed Yield
The current lesquerella seed yields are approximately 1800 kg/ha, but it is felt
that the plant has the potential of yielding 2500–3000 kg/ha. This increase will
come through a combination of improved agronomic practice and breeding.
Some agronomic issues include more precise plant spacing, better irrigation
management, and more efficient harvesting. Developing more productive vari-
eties will include selection based on harvest index and for specific environments.
L. fendleri is the most productive of all species so far tested because of its
extensive branching and subsequent flowering along each branch. Selection
for branching at warmer or cooler temperatures will identify plants better
adapted for growth in different climates.
Plants that reach maturity in a shorter time period will save production costs.
This will require selection based on seed germination at cooler temperatures, or
plants that will branch at lower temperatures.
18.5.3 Wider Adaptation and Shorter Growing Period
Since these two goals are related, they will be discussed together. These issues
are also tied into the discussion above on improved seed yield. If plants are able
to be developed that branch at lower or cooler temperatures, the planting date
could be moved from October to February. Planting later than February in
Arizona is not desirable because of the probability of summer rains that may
occur during the dry down period causing seed shatter. Lesquerella does not
normally shatter; however, when irrigation is terminated and a desiccant is
applied 7–10 days before combining, the crop exposed to hard rains could lose
its seed yield.
18.5.4 Autofertility
A few Lesquerella species are autofertile such as L. pallida and L. mcvaughiana.

Both have the same chromosome number as L. fendleri with potential for use as
a source of germplasm to introgress this desirable trait. Within L. fendleri there
may also be potential for variation in autofertility, but it has not been looked for
in a thorough manner. The advantage of autofertility would be that pollinators
would not be required to increase seed yield. Plants may begin flowering in
February or earlier in warmer years but these temperatures are still too cool for
pollinators to be present in the field which causes a yield reduction. There are
also years when feral bee populations are not as abundant resulting in lower
yields, unless managed bees are used. The cost of bees for pollination obviously
results in higher production costs and can be significant due to problems
beekeepers are experiencing such as colony collapse disorder. Self pollination
might prove to be detrimental due to inbreeding depression, but the trait still
warrants investigation.
Lesquerella, as well as other oilseeds in the Brassicaceae family, are generally

cross pollinated and self incompatible. A single flower produces up to 30 seeds
inside a silique (pod) and each seed has the potential to originate from a
different pollen source causing each seed to be genetically different. Although
more genetic variability for selection is generated, a desired trait is more
difficult to select because the seeds from a single plant are half instead of full
siblings, as self pollination rarely occurs (Dierig et al. 2004). Half seed selec-
tions and inheritance studies have allowed selection of mutants in the fatty acid
profile instead of through half sib selection. The ability to analyze a half or a
single seed of L. fendleri for fatty acid and oil content has greatly improved
breeding of this crop (Isbell et al. 2008). Since the seed is very small, this has
proved to be a greater challenge compared to other oilseed crops where it has
been accomplished. The seed of L. fendleri weighs about 0.6 mg, which is less
than half the size of alfalfa seed.
Interspecific hybrids have been a focus of our program to utilize traits
not found in L. fendleri but that occur in other Lesquerella species. Eastern
species hybridize in nature but species native to the western U.S. do not
hybridize readily. The current method used for producing these hybrids is
described in Dierig et al. (2004). This includes use of ovule culture and
colchicine treatment. Cytological information has also been essential to
determining the behavior of parents and progeny in these crosses. One
example of this was in a cross of another species with L. fenderi that was
thought to have the same chromosome number. When some of the
progeny failed to have the high HFA trait of this parent, cytological
examination, using pollen mother cells, found that the seed which had
originated from an exploration trip and was thought to be a single species,
was actually a mix of two different species (D.T. Ray, personal data). Bud
pollinations, where buds are manually opened before anthesis to pollinate,
are routinely used in both inter- and intra-specific crosses to overcome
incompatibility. Other breeding methods include those used for cross
pollinated crops such as recurrent selection.
Mutation breeding has been utilized in our program using ethyl
methane sulfonate (EMS). We have found that L. fendleri has a tremen-
dous amount of variability and many of the mutants we have in our
collection such as fatty acid mutants (0% lesquerolic acid + high oleic;
0% linolenic; 0% linoleic), cream flower color, male sterility, yellow seed
coat; a 5-petal flower, and multi-locule silique, also occurred without EMS
treatment.
18.7 Integration of Information Technology

and New Biotechnologies
Information technology has aided the progress of lesquerella breeding by

integrating data management and creating a more efficient use of plant germ-
plasm. White et al. (2007) described the importance of this especially with new
crop development where the scientific input is much reduced compared to
established, traditional crops. New crop development is especially prone to
fluctuating efforts over time due to the limited available financial resources.
Often times research results do not get published because funding ends or a
researcher retires. Intellectual Property Rights can also be an issue for exclusive
licensing of products from new crops or Plant Variety Protection and germ-
plasm used in developing the variety along with breeding methods. The goal of
developing an information technology system for lesquerella has been the
documentation of germplasm and collection information, maintaining careful
records of genealogy and population structures, and developing a network
available to interested users. This information system keeps careful documenta-
tion of all the available phenotypic and molecular documentation. The system
we have utilized is the International Crop Information System
(ICIS, www.icis.org).
SSR markers have been developed for lesquerella (Salywon and Dierig 2006).
These SSRs along with other molecular markers have value in association
mapping of traits, such as lower linolenic acid content. The association mapping
methodology calculates linkage disequilibrium and is superior to quantitative
trait loci (QTLs) mapping in determining genetic variances (Zhao et al. 2007).
Bioinformatics approaches such as searching the EST libraries for DNA
sequences with similarity to important genes are now being used in lesquerella.
Necessary genes to incorporate HFA at the sn-2 position of L. fendleri seed oil
are being examined (Dyer and Mullen 2005).

There is currently no lesquerella commercial seed production. Technology
Crops International (TCI, www.techcrops.com) is currently working on a
marketing strategy to begin production. They plan beginning commercial
production of lesquerella seed in areas of the southwestern U.S.
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Barclay, A.S., Gentry, H.S. and Jones, Q. (1962) The search for new industrial crops II:
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Dierig, D.A., Salywon, A.M. and Tomasi, P.M. (2008) The inheritance of a high oleic – zero
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Dierig, D.A., Thompson, A.E., Rebman, J.B., Kleiman, R. and Phillips, B.S. (1996) Collec-
tion and evaluation of new Lesquerella and Physaria germplasm. Ind. Crop. Prod. 5,
53–63.
Dierig, D.A., Thompson, A.E. and Coffelt, T.A. (1998) Registration of three Lesquerella
fendleri germplasm lines selected of improved oil traits. Crop Sci. 38, 287.
Dierig, D.A., Tomasi, P.M. and Ray, D.T. (2001) Inheritance of male sterility in Lesquerella
fendleri. J. Amer. Soc. Hortic. Sci. 126, 738–743.
Dierig, D.A., Tomasi, P.M., Salywon, A.M. and Ray, D.T. (2004) Improvement in hydroxy
fatty acid seed oil content and other traits from interspecific hybrids of three Lesquerella
species; Lesquerella fendleri, L. pallida, and L. lindheimeri. Euphytica 139, 199–206.
Dierig, D.A., Tomasi, P.M., Salywon, A.M., Dahlquist, G.H. and Isbell, T.A. (2006b) Varia-
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Dyer, J.M. and Mullen, R.T. (2005) Development and potential of genetically engineered
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Goodrum, J. and Geller, D. (2005) Influence of fatty acid methyl esters from hydroxylated
vegetable oils on diesel fuel lubricity. Bio. Res. Tech. 96, 851–855.
Hayes, D.G., Kleiman, R. and Phillips, B. (1995) The triglyceride composition, structure, and
presence of estolides in the oils of Lesquerella and related species. J. Am. Oil Chem. Soc.
72, 559–565.
Isbell, T.A., Mund, M.S., Evangelista, R.L. and Dierig, D.A. (2008) Method for analysis of
fatty acid distribution and oil content on a single Lesquerella fendleri seed. Ind. Crops
Prod. 28:231–236.
Jenderek, M.M., Dierig, D.A. and Isbell, T.A. (2008) Fatty acid profile of Lesquerella
germplasm in the National Plant Germplasm System collection. Ind. Crops Prod.
29:154–164.
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(Cruciferae). Sida 10, 167–175.
O’Kane, S.L., Al-Shehbaz, I.A. and Turland, N.J. (1999) Proposal to conserve the name
Lesquerella against Physaria (Cruciferae). Taxon 48, 163–164.
O’Kane, Jr., S.L. (1999) Lesquerella navajoensis (Brassicaceae), a new species of the L. hitchcockii
complex from New Mexico. Madrono 46, 88–91.
O’Kane, S.L. and Al-Shehbaz, I.A. (2002) Paysonia, a new genus segregated from Lesquerella
(Brassicaceae). Novon 12, 379–381.
Payson, E.B. (1921) A monograph of the genus Lesquerella. Ann. Missouri Bot. Gard. 8, 103–236.
Roetheli, J.C., Carlson, K.D., Kleiman, R., Thompson, A.E., Dierig, D.A., Glaser, L.K.,
Blase, M.G. and Goodell, J. (1992) Lesquerella as a source of hydroxy fatty acids for
industrial products. USDA-CSRS, Office of Agricultural Materials. Growing Industrial
Materials Series. 46 pp.
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57, 241–264.
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Harvard Press, Cambridge, MA USA.
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Rollins, R.C. (1997) Two Lesquerellas (Cruciferae) of south central and western Montana.
Novon 5, 71–75.
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(Cruciferae) from the State of Washington. Rhodora 97, 201–207.
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Lesquerella fendleri (Brassicaceae) and cross-species amplification. Mol. Ecol. Notes 6,
382–384.
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new Lesquerella and Physaria (Brassicaceae) oilseed germplasm. Am. J. Bot. 92, 53–62.
Smith, Jr., C.R., Wilson, T.L., Miwa, T.K., Zobel, H., Lohmar, R.L. and Wolff, I.A. (1961)
Lesquerolic acid. A new hydroxy acid from Lesquerella seed oil. J. Org. Chem. 26,
2903–2905.
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a new industrial oilseed. Ind. Crops Prod. 2, 97–106.
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develop new industrial crop cultivars. In: H.L. Shands and L.E. Weisner (Eds.), Use of
Plant Introductions in Cultivar Development, Part 2. CSSA Special Publication No. 20.
Madison, WI, pp. 9–48.
White, J.W., Dierig, D.A., Tomasi, P.M., Salywon, A.M. and Nath, D. (2007) Harnes-
sing information technologies for more efficient crop development. In: J. Janick and
A. Wipkey(Eds.), Issues in New Crops and New Uses. ASHS Press, Alexandria, VA.
USA, pp. 8–18.
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D. and Koornneef, M. (2007) Association mapping of leaf traits, flowering time, and
phytate content in Brassica rapa. Genome 50, 963–973.
Chapter 19
Cuphea
Winthrop B. Phippen
19.1 Introduction
Temperate plant species whose seed oils are rich in medium-chain fatty acids
(MCFAs) are relatively rare (Wolf et al. 1983). One species of particular interest
is cuphea, a temperate annual oilseed crop with high levels of MCFAs such as
capric and lauric acid (Graham et al. 1981; Graham and Kleiman 1985;
Graham and Kleiman 1992). These fatty acids are highly valued as feedstocks
in manufacturing cosmetics, soaps and detergents, pharmaceuticals, and indus-
trial lubricants (Wolf et al. 1983; Thompson et al. 1990; Cermak and Isbell
2004). Additionally, new uses for MCFAs have the potential to significantly
replace petroleum-based products like motor oil, hydraulic fluid, and diesel fuel
(Geller et al. 1999; Geller and Goodrum 2000; Leroux et al. 2006). This chapter
primarily covers the advances of cuphea research since the previous reviews of
cuphea breeding presented by Knapp (1990a, 1993) with the main focus on
oilseed production in the cuphea species of C. lanceolata, and C. visscosissima
which are targeted for commercialization in the US.
Research interests have centered on cuphea primarily for its seeds which
have a high content of unique fatty acids (Earle et al. 1960; Graham et al. 1981;
Wilson et al. 1960). The predominant fatty acids include: caprylic acid (C8),
capric acid (C10), lauric acid (C12) and myristic acid (C14). Lauric acid has
been the primary fatty acid of interest in US breeding programs. Lauric acid is
used in foods, mostly vegetable shortenings, and in soaps and detergents as a
defoaming agent and booster (Thompson 1984; Babayan 1981). Tradition-
ally, the tropical oil crops such as coconut (Cocos nucifera L.) and oil palm
(Elaeis guineensis Jacq.) commercially supply these acids. Currently the US
soap and detergent industry gets half of these fatty acids from the petroleum
industry and the other half from imported coconut and palm kernel oils
(Hardin 1991). Coconut oil is 45–50% lauric acid, while some undeveloped
lines of cuphea can produce oil that contains nearly 80% lauric acid (Graham
W.B. Phippen (*)

Department of Agriculture, Western Illinois University, Macomb, IL, USA
e-mail: WB-Phippen@wiu.edu

518 W.B. Phippen
1989b). To date, there are no temperate oilseed crops that can supply these
lipids (Ignacio 1985; Arkcoll 1988). Many of the fatty acid-rich species of
cuphea are summer annuals and through crop development programs could
potentially become domestic sources of fatty acids.
The cuphea breeding line, ‘PSR23’ (Cuphea viscosissima Jacq. C. lanceo-
lata W.T. Aiton) is the only domesticated cuphea in the US and is the current
focus of production and breeding programs in Illinois, Iowa, North Dakota,
and Minnesota. Although rich in capric acid, PSR23 serves well as an
agronomically sound plant for agronomic and physiological studies and as a
foundation for breeding programs in the US.
Breeding and research efforts on other Cuphea spp. continues throughout the
world. Research in India is focused primarily on C. procumbens for seed oil
production (Pandey et al. 2000; Rameshkumar et al. 2002; Singh and Singh
2002; Singh and Rameshkumar 2003; Singh et al. 2007). South American
researchers are looking at C. carthagenesis (Mathioni et al. 2005; Dezanet
et al. 2007) and C. glutinosa (Yagueddu et al. 2006) for medicinal purposes.
Japanese researchers are investigating food uses with C. leptopoda (Saikusa
et al. 2001).
19.2 Domestication and Breeding History
The genus Cuphea (Lythraceae) contains approximately 260 species that are
native to the area from Mexico through Brazil, with one species native to the
eastern United States (Graham and Kleiman 1985). Several are adapted to
temperate agriculture and have seed oils rich in capric and lauric acid. Initial
domestication programs began in Germany to evaluate different species and to
determine the feasibility of domesticating cuphea (Hirsinger 1980; Hirsinger and
Röbbelen 1980; Graham et al. 1981; Hirsinger and Knowles 1984; Hirsinger 1985;
Röbbelen and von Witzke 1989). At Oregon State University (OSU), Steven
Knapp began an intensive breeding program to domesticate cuphea for US
production. Knapp worked extensively with C. viscosissima, the only species
native to the United States, and C. lanceolata, a species native to the Sierra
Madre of Mexico (Graham 1988). The essential agronomic traits and seed oil
yields of these species were sufficient for competition as oilseed crops in the US.
Seed shattering and seed dormancy were the major domestication barriers within
the genus at the time (Knapp 1990a,b).
In the mid to late 1990s, important breakthroughs were made towards
eliminating seed shattering and seed dormancy by exploiting interspecific
diversity. Non-shattering phenotypes within the C. viscosissima C. lanceolata
f. silenoides population and an autofertile, non-dormant, and non-shattering
C. viscosissima C. lanceolata f. silenoides cultivar have all been developed
(Knapp and Crane 2000a,b,c). Knapp was also successful at developing other
19 Cuphea 519
elite lines of cuphea that are non-shattering, auto-fertile, and have reduced
levels of sticky hairs.
Sticky or glandular hairs covering stems, leaves, and flowers are characteristic
of most cuphea species (Graham 1988; Amarasinghe et al. 1991). These glandular
hairs have been cited as a negative trait primarily due to the difficulty in harvest-
ing the crop (Hirsinger 1980; Hirsinger and Röbbelen 1980; Thompson 1984).
The stickiness of cuphea is certainly difficult when working by hand, but it is not a
limitation to the commercialization of cuphea. Recent large scale production of
several hundred acres in 2005–2007 in Minnesota and Illinois were not impeded
by the stickiness when production combines were used (Fig. 19.1). Although
some of the sticky residue from cuphea chaff accumulates in harvesting equip-
ment, it does not seem to hinder harvesting. The indeterminate nature, high levels
of moisture, and shattering levels of cuphea are seen as the current barriers to
successful and consistent harvests.
Fig. 19.1 Direct combining of PSR23 cuphea in Illinois
19.2.1 Oil Crop Breeding
Studies conducted in the early 1980s on cuphea focused primarily on character-

izing fatty acid profiles of various cuphea species collected from the wild
(Graham et al. 1981; Wolf et al. 1983; Hirsinger 1985). These studies helped
direct early breeding programs to focus on lauric acid accumulating species,
C. wrightii, for the soap and detergent industry (Thompson and Kleiman 1988).
Unfortunately, domestication of this species proved to be extremely difficult
520 W.B. Phippen
due to seed shattering, open pollination, and poor agronomic traits. However,
recent efforts with the high capric acid species of C. viscosissima and
C. lanceolata have been successful in improving many of the agronomic traits
necessary for cuphea domestication (Knapp 1993). Several early breeding
programs employed mutation breeding to address fatty acid accumulation
(Hirsinger 1980; Hirsinger and Röbbelen 1980; Campbell 1987; Röbbelen and
von Witzke 1989; National Botanical Research Institute 2003); however, they
all experienced limited success. No newer reports of mutation breeding have
been reported in the literature. Non-shattering and determinacy still pose
difficult hurdles in cuphea species. To date, variety trails of cuphea accessions
have yet to identify a completely non-shattering or determinate accession. With
efforts continuing on improving agronomic traits, altering total oil and fatty
acid content will soon become a priority once again.

The USDA-ARS National Plant Germplasm System (NPGS) cuphea collection
is currently maintained at the North Central Regional Plant Introduction
Station (NCRPIS) in Ames, IA under the curation of L. Marek. As of January
2008, 649 accessions of cuphea are in the NCRPIS collection; however, only 499
are available through the Germplasm Resources Information Network (GRIN)
for distribution (a few as vegetative cuttings only, the rest as seed only). The
accessions represent 67 different taxa, plus seven accessions that have not been
identified to species, and 30 accessions that are hybrids of various combina-
tions. The available accessions represent 38 different taxa, plus 2 unidentified to
species, and 26 that are hybrids.
Over the past 10 years, the NCRPIS has distributed approximately 4,250
cuphea items in 167 requests. Nearly 80% of all requests were sent within
the US. International distributions were to Japan, the Netherlands,
Germany, France, China, England, Brazil, Greece, and Canada. Cuphea
seed requests are distributed evenly between ornamental and oil related
uses (breeding, enzymes, development, and evaluation) (L. Marek, personal
communication).
The cuphea genetic resources collection was recently assisted by the identi-
fication of protocols to help remove dominancy issues of wild type accessions
(Widrlechner and Kovach 2000; Crane et al. 2006). It was also found that the
current storage protocols were detrimental to seed germination (Volk et al.
2006). C. wrightii A. Gray, C. laminuligera Koehne, C. carthagenensis (Jacq.)
J.F. Macbr., and C. aequipetala Cav are considered sensitive to low temperature
storage. The seeds of these species have triacylglycerols that are crystalline
at –188C and melt when the seeds are warmed to >358C (Volk et al. 2007).
Cuphea seeds imbibed while the triacylglycerols are crystalline fail to germinate
and exhibit visual damage. However, germination proceeded normally when
19 Cuphea 521
dry seeds were warmed adequately to melt any crystalline triacylglycerols

before imbibition.
Although most of the available accessions have basic descriptors, the major-
ity of the collection has limited information regarding fatty acid content and
yield. Several studies have screened portions of the collection, but very little
consistency exists between them (Graham et al. 1981; Wolf et al. 1983; Hirsinger
1985). Extraction method, location and year where seeds were produced, and
gas chromatography analysis have all led to variations in total oil and fatty acid
composition being reported for individual accessions. Many of the early studies
often involved complicated and difficult extraction and analysis procedures
requiring hours of preparation. These methods are impractical for supporting
high throughput breeding programs aimed at developing new varieties. A recent
study by Phippen et al. (2006) developed a reliable and efficient method for
determining total oil and fatty acid content in cuphea seed and evaluated 185
cuphea accessions.
19.4 Advances in Cuphea Production
Knapp, while at OSU, essentially maintained the only cuphea breeding

program in the US from the mid 1980s to 2004. Many of his breeding
advancements were summarized in Knapp (1993) and more recently in
Knapp et al. (2004).
Knapp’s more recent efforts were directed towards: the development of fully
self-pollinated, partially non-shattering cultivars; breeding for high oil, high
capric acid, and semi-determinant flowering; and strategies for developing high
lauric and fully non-shattering cultivars (Knapp et al. 2004). The development
of high capric cultivars is underway using high capric acid (85–89% C10)
germplasm sources. As reported by Knapp et al. (2004), the molecular breeding
program was restarted to build the foundation for genetic analyses and marker-
assisted selection in cuphea. More than 200 sequence-tagged-site (STS) markers
have been developed. The molecular genetic diversity in C. viscosissima and
C. lanceolata has already been surveyed, along with the development of segre-
gating populations and near-isogenic lines for mapping and manipulating
phenotypic loci. Quantitative trait loci for seed dormancy, seed shattering,
self-pollination, and other economically important traits continue to be identi-
fied. This advancement effort is aided by a genetic map of cuphea developed by
Webb et al. (1992).
19.4.1 ‘PSR23’ Cuphea

One of the first successful releases of a cuphea breeding line was ‘PSR23’ (PI
606544) released by Knapp and Crane (2000c). PSR23 was the first line to
522 W.B. Phippen
introduce the critical ‘Partial Shatter Reduction’ (PSR) trait into cuphea. This
trait inhibits the rotation of the placenta from the capsule thereby leading to an
increase in seed retention (Fig. 19.2). Typical wild type cuphea populations have
a near 100% seed loss due to shattering. The PSR trait reduces this seed loss to
only 20–30%. Other traits of interest in PSR23 include relatively high oil
content of 295 g kg1, high capric acid content of 72%, self-compatibility,
and non-dormant seed. Although still largely open-pollinated, PSR23 was
selected as the first cuphea line to begin commercialization.
Fig. 19.2 Partial shatter reduction trait in PSR23 cuphea with reduced placenta rotation (A).
Wild type shattering trait with increased placenta rotation (B)
PSR23 was first distributed to research programs in Illinois (IL) and Min-
nesota (MN) in the summer of 2000. The original population was heteroge-
neous and clearly demonstrated potential for environmental selection. Over the
next 7 years, selections were increased in IL and MN, remained separated and
developed unique phenotypes for each region. Under the drier and longer IL
growing season, a larger, erect, and less vegetatively pronounced phenotype was
favored. Unfortunately, this phenotype lost the essential PSR trait. In the
cooler shorter season of the northern corn belt, a smaller, more vegetatively
pronounced, and more compact phenotype prevailed. With this phenotype, the
reduced shattering trait remains intact.
A recent environmental adaptation study of the PSR23 ecotypes was con-
ducted in IL and MN in 2007. Preliminary results from the Illinois field studies
indicate that IL grown PSR23 selected in 2006 produced the least amount of
seed in 2007 (210 kg ha1), which is not surprising due to the lack of the PSR
trait. However, due to the small plant size of the MN grown PSR23 selected in
2006 its seed production under IL growing conditions was limited to 240 kg
ha1. When compared to the original PSR23 with a seed yield of 350 kg ha1,
both the MN and IL ecotypes fared worse under IL conditions. Under MN
growing conditions, all cuphea lines performed much better than in IL with the
original PSR23 reporting 830 kg ha1. Interestingly, only the first year selection
in MN performed as well as the original line. After 7 years of selection at both
19 Cuphea 523
locations, most lines significantly decreased in seed yield when compared to the
original line. This suggests the original PSR23 is still heterogeneous and that the
current selection protocols are not adequate for maintaining high seed yields.
19.4.2 Commercialization
With PSR23 being the only cuphea line available in adequate volumes, many of
the more recent agronomic, plant physiological, and product development
research reports in the US are based on this line. Although two ecotypes of
PSR23 exist, they do not differ significantly in total oil yield and fatty acid
constituents.
This first major breakthrough in producing cuphea on a large scale was the
identification of herbicides that could control broadleaf weeds in production
fields. Fortunately, cuphea demonstrates tolerance to several soil-applied her-
bicides including ethalfluralin, isoxaflutole, and trifluralin, and one
postemergence herbicide, mesotrione (Forcella et al. 2005a). Repeated studies
in IL and MN have enabled researchers to secure a registered 24C herbicide
label for the mesotrione herbicide for application on cuphea in 2005. This
helped facilitate large scale seed increases by contracted producers. To help
control biennial wormwood (Artemisia biennis) and Canada thistle (Cirsium
arvense) in MN, clopyralid could also be used safely in conjunction with soil-
applied isoxaflutole (Papiernik et al. 2006).
The summer of 2004 marked the first year for an experimental commercia-
lization trial focused on developing an agricultural management strategy for
cuphea utilizing conventional technologies to minimize the need for specialized
equipment. Technology Crops International, in cooperation with the USDA
Agricultural Research Service, grew 18.6 ha of PSR23 within a 32 km radius of
Morris, Minnesota (45.358N, 95.538W). Although not all hectarage was suc-
cessful, the harvestable plantings produced seed yields ranging from approxi-
mately 78–744 kg ha1 at 12% moisture (Gesch et al. 2006). Being the first large
scale production of a partially domesticated breeding line, valuable knowledge
was learned through this experience that might not have been gained by plot-
scale experiments alone. PSR23 remains indeterminate displaying a wide range
of seed maturities at time of harvest. Gesch et al. (2006) indicated post-harvest
management of seed on a large-scale (e.g., drying, cleaning, and storing) was
problematic, suggesting further need for introducing determinacy into the
current cuphea breeding lines.
Much of the success of the 2004 commercialization trial was due to the many
research projects focused on improving the cultural practices of producing
cuphea. Recent studies have addressed seed germination response to
temperature (Berti and Johnson 2008), row spacing (Gesch et al. 2003; Sharratt
and Gesch 2004), sowing dates (Gesch et al. 2002; Forcella et al. 2005b),
temperature sensitivity (Gesch and Forcella 2007), nutrient requirements
524 W.B. Phippen
(Olness et al. 2005), irrigation studies (Gesch et al. 2004), seed physiological
maturity (Berti et al. 2007) seed drying (Cermak et al. 2005) and harvesting
methods (Berti et al. 2005; Forcella et al. 2007). Tisserat et al. (2008) have
indicated that applications of ultra-high CO2 treatments accelerated cuphea
PSR23 growth and development and aided in seedling establishment. The basic
knowledge gained by the previously mentioned experiments has been the single
greatest advancement towards breeding and ultimately commercializing
cuphea. Although significant progress has been made in production protocols
for cuphea, the lack of any significant breeding effort to develop auto-fertile,
non-shattering, and determinate plant lines still limit the success of cuphea as a
commercial crop.
19.4.3 Product Development
With the success of the 2004 commercialization trial and the following growing
seasons, several thousand pounds of seed were made available to the USDA-
ARS in Peoria, IL for product development. Some of the more recent advances
can be found in modifying the fatty acids of cuphea to meet current industrial
needs as lubricants (Evangelista and Cermak 2007), fuels (Leroux et al. 2006)
and cosmetics (Cermak et al. 2007).
A recent study even characterized the proteins in cuphea (PSR23) seed to
provide fundamental information on their size, amino acid profile, solubility
classes, and solubility behavior (Evangelista et al. 2006). The seed contained
32% (dry basis, db) oil and 21% (db) crude protein. Glutelins and albumins
accounted for 83.5% and 15.4%, respectively, of the total protein extracted.
PSR23 has been further investigated to determine the effects of oil processing
conditions on functional properties of the seed proteins to evaluate their poten-
tial for value-added uses (Hojilla-Evangelista and Evangelista 2006).
19.5 Current Breeding Goals

19.5.1 Lauric Acid Accumulation
The original US cuphea breeding programs focused on cuphea species rich in

capric acid, with the intent of crossing in lauric acid once an agronomically
sound plant had been obtained. Current breeding lines are all progeny of
C. viscosissima C. lanceolata f. silenoides, which are diploid with six chromo-
somes (n ¼ 6) and rich in capric acid. Attempts have been made in the past to
create high lauric acid accumulating cuphea types, however, much of this early
work was on non-adapted cuphea species. Koehne (1903) described five cuphea
interspecific hybrids that were documented from herbarium samples in Europe.
Röbbelen and Hirsinger (1982) observed spontaneous outcrossing among
19 Cuphea 525
species in their collection and were able to produce colchicine-induced hybrids

of several cuphea species. However, the only successful crosses were between
species with similar fatty acid profiles. Lorey and Röbbelen (1984) were also
able to create hybrids, but lacked any success in altering fatty acid content.
More recently, Ray et al. (1988) developed 18 interspecific hybrids between
eight different species by hand emasculation and controlled pollination. They
are the first to report a successful cross between a capric and lauric acid
accumulating cuphea species. Unfortunately, the crosses between C. leptopoda
and C. llaminuigera were conducted on species not suitable for commercial
production in the US. These results demonstrate the feasibility of developing
a lauric acid accumulating interspecific hybrid using the capric species.
In 2001, a new cuphea breeding program was established at Western Illinois
University (WIU) to address the introgression of lauric acid accumulation into
the progeny lines for PSR23. The breeding program was initially supported by
private industry and was highly encouraged to only employ traditional breeding
methods for fear of losing certain commercial markets. This approach was
supported by the promising results from Ray et al. (1988) which suggested a
more traditional approach was warranted to help create an agronomically
sound high lauric cuphea plant.
Cuphea species have a tremendous variation in chromosome numbers and
ploidy levels (Graham and Cavalcanti 2001). The original PSR23 is an auto-
gamous diploid with six chromosomes (n ¼ 6). Selected wild accessions sui-
table for agronomic production are mostly diploid, but vary in number
of chromosomes from 8 to 14. C. lutea (n ¼ 14), C. viscosissima (n ¼ 6),
C. tolucana (n ¼ 12), C. wrightii (n ¼ 22), and C. carthagenesis (n = 8) are
the autogamous species (Graham 1989a). C. lutea and C. wrightii are
undoubtedly allotetraploids (Campbell 1987).
To help overcome the barriers in creating hybrids between the wild acces-
sions and the PSR23 line, various breeding methods are being employed. Initial
inter-specific crosses were conducted between PSR23 (PI 606544) and C. lutea
accessions utilizing hand emasculation techniques and pollen mixing proce-
dures as described by Ray et al. (1988) and Fehr (1987). Thirty two crosses
were successful and were evaluated in the field in 2005. From these crosses, four
progeny lines were selected for the 2006 growing season. Fatty acid profiles of
these progeny indicated a slight increase in lauric acid (C12) from 2.9 to 4.9%,
but a much larger increase in C14 (2.9–23.5%) and C16 (4.1–11.9%). Unfortu-
nately, all four progeny lines were unstable and reverted to the original profiles.
To improve success of the interspecific crosses, seedlings of PSR23 were
treated with a meiotic inhibitor, colchicine, to create colchiploids. The use of
colchicine and other meiotic disruption chemicals have been shown to be
successful in creating hybrids in cuphea and other species (Przybecki et al.
2001; NBRI 2003). Large scale plantings of PSR23 are mainly pollinated by
resident bumblebees. The long floral tubes prevent honeybees from gaining
access to the nectar. Commercial production of PSR23 will remain limited
without solving the insect pollinator problem. It is hypothesized that by
526 W.B. Phippen
increasing the ploidy level of the PSR23 not only will self-fertilization be
increased (Barringer 2007), but the chances of developing interspecific crosses
with the high lauric acid accessions would be improved. Successful ploidy level
changes were identified by increased vigor and cytologically confirmed using
pollen mother cell analysis techniques as described by Ray et al. (1989). Con-
firmed polyploid PSR23 plants were found to be fertile and were later crossed
with high lauric acid accumulating accessions of C. lutea, C. tolucana, and
C. carthagenesis in 2006. Seeds collected from each cross were excised and
germinated to avoid any seed dormancy issues (Mathias et al. 1990). S1 progeny
were evaluated under field conditions in 2007. Fatty acid profiles indicated once
again a significant increase in myristic acid (C14) with no increase of lauric acid
(C12). Selection criteria for the S2 progeny will be for self-pollination and
increased lauric acid content. Hybrids with the highest seed and oil yield will
be further selected to improve seed weight and total yield in subsequent years.
Along with developing lauric acid accumulating PSR23 progenies, the WIU
program continually focuses on developing new varieties utilizing mass and
recurrent selection for improving agronomic traits including increased vigor,
stem rigidity, drought tolerance, and self-pollination.
19.5.2 Insect Resistance

With cuphea production limited in the US to the Midwest and upper Midwest,
the adaptability of cuphea to the current crop rotation and production proto-
cols of the region play a critical role in the success of growing this new crop.
Proposing cuphea as a new broadleaf crop may provide an undesirable habitat
for corn, soybean, and wheat pests. Research conducted by the USDA-ARS
recently investigated the potential of utilizing cuphea in crop rotations to
provide cultural control of Western corn rootworm beetles (Diabrotica virgifera
virgifera LeConte). It was hypothesized that cuphea, because of its sticky sur-
face, would reduce or prevent oviposition in these fields. Unfortunately after an
intensive 4 year study with seven rotation programs, it was concluded that a
crop rotation with cuphea would not provide any consistent, economical cul-
tural control of corn rootworm (Behle and Isbell 2005).
From 2000–2005, no major insect pests were recorded as feeding on cuphea
vegetation. The only known insect pests were flea beetles (Altica spp.) which
could cause early season damage to young seedlings if not controlled. During
the 2006 growing season, elevated corn earworm (Helicoverpa zea Boddie)
populations were experienced in much of the Midwest including cuphea pro-
duction research sites in central and west-central Illinois. Corn earworm larvae
were observed feeding on cuphea leaves during August and early September
and feeding shifted to flowers and seed pods as larvae counts increased through
September. By crop harvest time in October, seed losses to corn earworm in IL
were estimated at 90%, while sites elsewhere in the state experienced more than
19 Cuphea 527
50% seed loss. Minor losses were reported in Iowa, with only occasional
sightings occurring in North Dakota, and no presence was reported in
Minnesota. Effective insecticides are available, but generally require repeated
applications and entail substantial monetary and environmental costs. Use of
resistant crop varieties is generally acknowledged as the best core strategy for
avoidance of corn earworm damage.
Evaluation plots of novel cuphea genotypes located adjacent to production
fields in IL displayed a wide range of severity and timing of feeding losses;
suggesting that genetic sources of elevated corn earworm feeding resistance may
be available. A 2007 breeding project evaluated forty accessions representing 16
different species of cuphea for reduced larval feeding. The accessions were
selected based on their agronomic potential in the Midwest, self-pollination,
and diverse sticky hair structures. Preliminary results indicate larval insects
have a preference for certain cuphea species and that several species demon-
strated no larval feeding damage throughout the entire growing season. It is
believed that the variations in the sticky hairs are contributing to the effective
defense against insect pests. Aphids and many other insects are typically immo-
bilized by the sticky hairs. A breeding program is already initiated to cross the
identified resistant accessions with the original PSR23 line. In contrast, non-
sticky mutants have been reported for C. lanceolata (Hirsinger 1980; Hirsinger
and Röbbelen 1980; Knapp 1993). Although a non-sticky trait might prove
useful in aiding harvest, the role of sticky hairs as a defense mechanism certainly
outweighs the convenience in handling the crop.
19.5.3 Anthocyanin Mutants
During the summer of 2004, variations in flower pigments were noticed in

PSR23 production fields in both MN and IL. PSR23 is characterized by deep
purple flowers with red pigmented stems. Several novel plants were collected
including a completely all white flower and variations of pink flowers.
The all white flower phenotype is devoid of any anthocyanin or red pigments
throughout the entire plant (Fig. 19.3). Even under cold and nutrient stress
conditions, the all white phenotype will remain. Named as ‘Snowflake’ in IL
and ‘Blizzard’ in MN, both all-white lines are exhibiting inbreeding depression.
After five self-pollinated generations in a growth chamber, the anthocyaninless
phenotype appears to be stable. However, this line clearly demonstrates
diminished vigor and seed set after each cross. When grown under field condi-
tions, Snowflake performs well early in the growing season but soon collapses
under environmental stress. Anthocyanins have been shown to serve as
antioxidants and enable plants to better deal with environmental stresses
(Chalker 1999). Efforts are currently under way to backcross Snowflake to
the original PSR23 to develop an all white flower plant with anthocyanin
production limited to the vegetative tissue. The all white flower is not only
528 W.B. Phippen
‘Snowflake’ ‘PSR23’
Fig. 19.3 Comparison of the all-white ‘Snowflake’ breeding line flower (A) and stem (C) to the
anthocyanin rich ‘PSR23’ breeding line flower (B) and stem (D)
19 Cuphea 529
unique for ornamental applications but also appears to have improved seed
retention and produces seed coats devoid of anthocyanin pigments. This could
potentially play a role in developing seed oils with less pigment contamination,
thus diminishing production costs.
19.6 Breeding Methods
19.6.1 Genetic Engineering

As with the development of any new crop, the time required to domesticate and
develop new varieties utilizing traditional methods is extremely slow. Several
research programs have investigated developing tissue culture methodologies
for propagation and perhaps engineering new traits. Early work focused on
explant propagation methodologies for C. wrightii (Janick and Whipkey 1986)
and C. tolucana (Przybecki et al. 2001). Other studies began screening a wider
variety of species for the potential of utilizing engineering techniques (Millam
et al. 1997). However, it is currently unrealistic to pursue genetically engineering
cuphea for large scale agronomic production. The most likely arena cuphea can
serve is as a rich source of genes encoding enzymes specialized for seed-specific
synthesis of short- and medium-chain fatty acids (Filichkin et al. 2006). If
cuphea species fail to be commercialized, they can still potentially provide a
diverse source of seed-specific genes for manipulating fatty acid content in other
oilseed plants.

Although the advancement of cuphea breeding has been slow, tremendous
progress has been made in developing the basic understanding of cuphea
physiology and agronomic production guidelines. The identification and devel-
opment of new potential industrial products and biofuels from cuphea oils are
also helping to increase the awareness of this unique oilseed crop. However, for
cuphea to truly be successful as a new commercial crop grown on large scale
volumes, continued industry support and the development of auto-fertile, non-
shattering, and determinate plant lines are needed. I would certainly like to
invite fellow breeders to take up the challenge and assist in advancing the
breeding efforts to locate or create these much needed traits.
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Index
A B
Accession numbers of crops in genebank, 7 Bacterial blight by Xanthomonas campestris,
Aceria guerreronis, 381 cotton diseases, 268
Aerobic desaturase/elongase-type Barnea and Basento cultivars, 408
pathway, 38 See also Olive
Aflatoxin by Aspergillus spp. in peanuts, 288 BC-2 Cultivars, 463
All India Coordinated Research Project on See also Poppy seeds
Oilseeds (AICORPO), 429 Benign prostate hyperplasia (BPH),
All Russian Flax Research Institute, 247 474–475
Alternaria blight by Alternaria spp. Best linear unbiased prediction (BLUP)
in oil seed rape, 135 technique, 346–347
in sunflower, 164 Biofuels production from vegetable
Ametiszt cultivars, 463 oils, 92
See also Poppy seeds Bison cultivar, 252
Amplified consensus genetic markers See also Flaxseed
(AGGM) in B. napus genetic Blackleg by Leptosphaeria maculans, 135
maps, 113 resistance by B. napus, 96
Anaerobic polyketide synthase (PKS) Bladderpod, 508
pathway, 38 Blue disease for cotton, 268
Annual world oil crop production, 1 Boll rot for cotton, 268
Anthocyanin mutants BrasEDB website for agronomical
all-white snowflake breeding line flower, evaluation data, 95
comparison of, 528–529 Brassica spp.
PSR23 production, flower pigments agronomic evaluation, 139–140
noticed in, 527 Brassica accessions from European gene
Applied Agricultural Resources Sdn Bhd banks, 94–95
(AAR) method, 350–351 Brassica napus as oilseed crop, 127
Arabidopsis-Brassica comparative genome breeding achievement in
analysis, 117 meal quality, 132
Arachidonic acid (ARA), 35 oil quality, 131–132
Arno cultivars, 408 current goals of breeding
See also Olive disease resistance, 135
Asian landraces, 65 fatty acid composition, 134
See also Soybean insect resistance, 135–136
Asian Vegetable Research and Development male sterility, 133
Center (AVRDC), 63 phytates and phenolic compounds
Askal cultivars, 408 reduction, 135
See also Olive seed color variations, 134
Auricolic acid, 508 seed meal quality, 134–135
535
536 Index
Brassica spp. (cont.) C

seed yield and adaptation, Caenorhabditis elegans o-3 desaturase and
132–133 pork fat with ALA
self-compatibility, 133–134 concentration, 39
tocopherol content, 134 Campesterol in soybean oil, 84
vernalization requirements and Canada
flowering time, 133 commercial sunflower hybrids
genetic and germplasm resources, 130 production, 168
genetic markers and linkage maps, western solin seed quality, 243
140–141 Canola oils
germplasm characterization, 142 polyunsaturated fatty acids source, 37
hybrid cultivars, 138–139 Carotenoids, 473
marker assisted selection, 144 Carthamus tinctorius L., see Safflower seed
molecular mapping Castor seed, 317
disease resistance, 144 breeding achievements
glucosinolate content, 143 allergy, 323
male sterility, 143 fatty acid composition, 320–321
morphological and agronomic quantitative and qualitative traits,
traits, 143 323, 325–326
oil content and quality, 142–143 ricinine and ricin toxins, 322–323
seed color, 142 breeding methods and techniques
vernalization requirements and backcross method, sexual
flowering time, 143–144 hybridization, 328
naturally existing genetic variation, individual plant selection with
manipulation of, 136–137 progeny tests, 326–327
origin and domestication mass selection, 326
B. carinata,129 pedigree and bulk method, 327
B. juncea,128–129 recurrent selection, 328
B. rapa,128 single seed descent (SSD) method, 328
population cultivars and breeding of line, breeding programs, 328–329
137–138 genetic resources, 319
seed production, 145–146 germplasm collections, 320
seed quality evaluation, 140 origin and domestication
selfing and artificial hybridization, 139 capsule spines, phenotypic variation
self-pollination in, 129 for, 318
transgenic breeding, 145 production, historical summaries
vegetable oil from, 127 of, 319
wild-type, single-zero and double-zero phenotypic variation in
cultivars, 130 pod color, 324
winter and spring cultivars, 130 seed shape, size and color of, 325
See also Oilseed rape seed production, 329
Breeding populations of restricted origins varietial groups, 319
(BPROs ), 336 Charcoal rot by Macrophomina phaseolina
Bronowski spring cultivars, 93, 100 in sunflower, 164
See also Oilseed rape China
Bronze wilt in cottonseeds, 268 genetic diversity in annual wild soybean
Broomrape parasitic plant and sunflower collection in, 64
production, 164 G. max accessions, collection of, 63–64
BROP-1 cultivars, 463 Chiquitita cultivars, 409
See also Poppy seeds See also Olive
Brown girdling root rot complex by Coconut palm, 369–370
Rhizoctonia solani, 135 breeding achievements
Burling’s Mexican cotton, 262 drought breeding, 379–380
Index 537
dwarf dwarf hybrids, 379 pedigree method, 275

hybridization, 378 timeline for commercial transgenic
pollinated progenies of, 377–378 cotton variety development, 277
tall tall hybrids, crosses between, transgenic cultivars by backcross
378–379 method, 276
breeding, goals classification of, 259–261
Aceria guerreronis, 381 current goals of breeding
dwarf tall hybrids, production of, private companies, 273
380–381 state universities, 272–273
breeding methods and techniques, 381 USDA-Agricultural Research Service
inter-varietal hybridization, (USDA-ARS), 272
383–384 USDA, germplasm exploration trips,
mass selection, 382 271–272
plus palm, 382–383 domestication of, 261–263
breeding programs flower with male and female parts, 259
genetic diversity analysis, 384–385 genetic resources, 264
genetic relatedness, 385–387 collection of, 265–266
hybridity testing and variety market opportunities for, 258
identification, 388 origin and domestication, 258
linkage mapping and QTL as protein source, 257
identification, 388–389 seed production, 279–280
somoclonal variation in, 388 use of, 257
synteny studies, 389 varietal groups, 263–264
in vitro culture, 390 Crop Development Centre (CDC) by flax
genetic resources, 375 breeding program, 240
Coconut Genetic Resources Network Cross-pollinated self-incompatible forms of
(COGENT), 376 B. rapa, 138
origin and domestication, 370–372 Crude palm oil (CPO), 333
seed production, 390 Cucurbita spp., 469–470
varietal groups, 372, 374–375 Cuphea spp.
tall and dwarf coconuts, contrasting breeding methods
features of, 373 genetic engineering, 529
Cocos nucifera L., see Coconut palm current breeding goals
Codominant markers in sunflower anthocyanin mutants, 527–529
breeders, 209 insect resistance, 526–527
Coincya monensis, 98 lauric acid accumulation, 524–526
Corn Belt Dent (CBD) domestication and breeding history
open-pollinated varieties (OPVs), 495 C. viscosissima C. lanceolata f.
Cottonseed silenoides cultivar, 518–519
breeding achievements oil crop breeding, 519–520
abiotic stress tolerance, 268–269 fatty acids in, 517
agronomic adaptation, 269 genetic resources, 520–521
fiber quality, 270–271 production
host plant resistance, 267–268 commercialization, 523–524
seed traits, 271 product development, 524
breeding methods and techniques PSR23 breeding line, 521–523
commercial cotton breeding, 276 Cylindrocladium black rot by
gossypol and precursors, breeders, 279 Cylindrocladium crotalariae in
insecticide and herbicide resistance, 278 peanuts, 288
marker-assisted selection, 278 Cytoplasmic male sterility (CMS)
mass selection within heterogeneous for Brassica crops, 108
population, 275 sunflower for, 162
modified agronomic traits in, 278 sources, 163–164
538 Index
D European sunflower moth, 166

Diacylglycerol acyltransferase Express widely-cultivated winter oilseed rape
(DGAT), 321 variety, 108
Dihomo-g-linolenic acid (DGLA), 39
Dihydrotestosterone (DHT), 474
Diversity Fixed Foundation Set (DFFS) for F
oilseed rape gene-pool, 95 FAD2-1 gene mutation and soybean 18:1
Docosapentaenoic acid (DPA), 43 concentration, 82
in EPA lines, 41 FAM94-41 USDA germplasm line, 82
Docosahexaenoic acid (DHA), 35 See also Soybean
ARA and EPA, precursors, 37 fanfan C1640 germplasm line, 83
consumption of, 37–38 See also Soybean
production in plants Fats consumption, 1–2
via aerobic elongation/desaturation Fatty acid
pathways, 43–44 content of plant oils modulation
via anaerobic polyketide synthase profile and oil functionality,
pathway, 44 31–32
Domestication dietary intake and overall health, 32
germplasm, 6 epoxidation of, 47
oil crops, 4 low-density lipoprotein (LDL) and
flax/linseed, 5 cardiovascular disease risk, 36
oilseed rape, 6 nomenclature and chemical structures, 36
sesame, 5 profiles in soybean derived from tools of
soybean, 6 biotechnology, 33–35
sunflower, 5–6 Flaxseed
Dominance theory, 496 breeding procedures
Downy mildew by Plasmopara halstedii backcross, 252
in sunflower, 164 bulk method, 252
Dumpy.AVROS variety, 339 combining parents methods, 249
breeding semidwarf high oil yielding doubled haploids, 252–253
palms, 340 pedigree method, 250–251
selection of parents, 248–249
single seed descent, 251
E cultivar development, 236
Early leaf spot by Cercospora arachidicola distribution of species in world, 236
Hori in peanuts, 288 germplasm sources, 247–248
Eckendorfer Blausamiger cultivars, 462 grain quality
See also Poppy seeds anti-nutritionals, 245–246
ECP/GR Central Crop Database for B. disease resistance, 246–247
napus accessions, 95 mucilage and lignan, 245
Eicosanoid biosynthesis for normal human oil and fatty acid composition,
physiology, 37 242–244
Eicosapentaenoic acid (EPA) protein, 244
EPA and/or DHA consumption, seed color, 246
cardiovascular disease and mental North American flax cultivars,
health, 37–38 237–239
production in plants origin and taxonomy, 235
via 6 desaturase pathway, 39–42 production of, 233
via 9 elongase pathway, 42–43 variety development objectives
Eicosatetraenoic acid (ETA), 39 grain yield, 241
Empire Cotton Growing Company, 261–262 lodging resistance, 241–242
European Cooperative Program for Plant maturity, 241
Genetic Resources (ECPGR), 130 world supply and disposition, 234
Index 539
FLOWERING LOCUS C (FLC) homologs International Board for Plant Genetic

role in flowering time and traits in Resources (IBPGR) guidelines for
B. napus, 115–116 safflower seed, 427
Full-sib hybrids in sunflower breeders, 183 International Olive Oil Council (IOOC)
data, 397
Interpopulation recurrent selection methods
G in sunflower breeders, 182–183
Garcinia mangostana, see Mangosteen seed Inter simple sequence repeat (ISSR) by
Genebank collections of Linum germplasm, 247 B. rapa, 141
Gene pyramiding in sunflower breeders, 210 Iowa State University (ISU), soybean lines
Genetic diversity in oil crops, 8–10 development of commercial use, 67
Genotypic recurrent selection methods in
sunflower breeders, 182
German genebank for flaxseed, 248 J
Germplasm Resources Information JA-16 cultivars, 463
Network (GRIN), 521 See also Poppy seeds
Global oil crop production and acreage, 2 Jet Neuf French cultivar, 97
Glycine max (L.) Merr., see Soybean See also Oilseed rape
GmFATB-1A gene deletion for oil
improvement in soybean, 81
See also Soybean K
Gossypium spp., see Cottonseed Kadesh cultivars, 408
See also Olive
Kék Duna and Kék Gemona cultivars, 463
H See also Poppy seeds
Hand emasculation in sunflower, 187 Kirtiman and Kompolti M cultivars, 463
Harvest index (HI) of pumpkin, 481 See also Poppy seeds
Helianthus annuus L., see Sunflower Kozmosz cultivars, 464
Heterosis, mechanism, 496 See also Poppy seeds
Heterotic group modelling in sunflower, 199
High-density linkage maps for B. rapa and B.
juncea,141 L
Hydroxy fatty acid (HFA) profile, 507–508, Late leaf spot by Cercosporidium personatum
510, 513 in peanuts, 288
Lauric acid accumulation
cuphea breeding program, 524–525
I PSR23, seedlings interspecific crosses,
Illinois high-oil/low-oil (IHO/ILO) long- 525–526
term selection experiment Lembkes cultivar, 92
maize kernel, chemical composition of, See also Oilseed rape
499–500 Lesquerella spp.
RLO and SHO population, 501 breeding achievements, 510–511
INDEL (Insertion-Deletion) markers in breeding methods and techniques, 513
sunflower, 197 current breeding goals
India autofertility, 512–513
safflower seed, hybridization of, oil content and fatty acid profile, 511
439–440 seed yields, 512
soybean production systems wider adaptation and shorter growing
Madhya Pradesh and on Malwan period, 512
plateau, 65–66 genetic resources
INRA-Ogura CMS system from radish, 100 L. pallida, breeding program, 510
Institute of Biodiversity Conservation and in North America, 509–510
Research (IBCR) of Ethiopia, 131 NPGS, 509
540 Index
Lesquerella spp. (cont.) inbred line development,

information technology and new 497–498
biotechnologies, integration overview of, 495
SSR markers and QTLs mapping recurrent selection (RS), 499
in, 514 Male Sterility Lembke (MSL) GMS system
Lesquerella fendleri plants, 511 for rapeseed hybrids, 100
origin and domestication, 507, 509 Mangosteen seed
flowers of, 508 and stearate contents, 34
seed production, 514 Marker-assisted backcross breeding in
Leukotrienes eicosanoid family of sunflower breeders, 209
metabolites synthesis from ARA Marker assisted selection (MAS)
and EPA, 37 in sunflower breeding programs
Liho spring cultivars, 93, 98 assays optimization and cost
See also Oilseed rape reduction, 208–210
Linoleic acid, 471–472 marker validation and refinement,
-linolenic acid (ALA) concentration in 206–207
diet, 39 Mendel winter oilseed rape varieties, 96
g-linolenic acid (GLA), 39 Michalko and Mieszko cultivars, 463
Linum spp., see Flaxseed See also Poppy seeds
Long-chain polyunsaturated fatty acids Modern maize breeding
(LCPUFA) production in germplasm
plants, 38 corn belt dent (CBD), 494–495
Long chain-o3 fatty acids in seed oils heterotic patterns, 495–496
engineering complex pathways into plant heterosis and inbred/hybrid concept,
seeds, 35 496–497
heterotic groups, 495–496
hybrid development, 498
M inbred line development
Maalot cultivar resistant to Spilocaea backcrossing, 498
oleagina, 408 marker assisted breeding, 497–498
Maharashtra Hybrid Seed Company overview of, 495
(MAHYCO), 429 Modified reciprocal recurrent selection
Mahndorfer cultivars, 462 (MRRS)
See also Poppy seeds breeding programs, 338–339
Maize, 493 Monaco cultivars, 463
high-oil, breeding programs See also Poppy seeds
for, 501 Mortierella alpina desaturase gene, 40–41
populations and commercial breeding Mozambique Tall
activities, 502 grouping of, 386
TopCross1and TC Blend1 seed corn Multi-headed restorer and single-headed
method, 503 maintainer population in
Illinois high-oil/low-oil (IHO/ILO) sunflower breeders, 183
long-term selection experiment, Mustard aphid pests of oilseed brassicas,
499–500 135–136
RLO and SHO population, 501
kernel structure and composition, 493
diagram of, 494 N
modern maize breeding National Bureau of Plant Genetic Resources
germplasm, 494–496 (NBPGR) of India, 131
heterosis and inbred/hybrid concept, National Germplasm Evaluation Program of
496–497 China (NGEPC), 63
heterotic groups, 495–496 National Plant Germplasm System (NPGS),
hybrid development, 498 509, 520
Index 541
National Research Centre for Soybean breeding programs, new biotechnologies,

(NRC Soybean), 65–66 integration of
National Soybean Germplasm Collection, 70 BASTA-resistant, 357–358
Nematodes by Meloidogyne spp. in clonal fidelity and performance
peanuts, 288 tests, 355
N78-2245 germplasm line, 82 clones, commercial planting of,
See also Soybean 354–355
N79-2077-12 germplasm line, 16:0 fluorescent in situ hybridization
soybean, 81 (FISH), 357
Nimbkar Agricultural Research Institute genetic markers, 356–357
(NARI), 429 molecular markers, 355–356
N.I. Vavilov Institute for Plant Industry, 247 polymorphic markers, 356
North Central Regional Plant Introduction tissue culture clonal propagation
Station (NCRPIS), 521 technique, 350–351
for sunflower germplasm, 160 tissue culture cloning process,
North Central Regional Plant Introduction 351–353
Station (NCRPIS) of USA, breeding techniques
130–131 breeding system and controlled
pollination, 347
seed germination, 347
O collections from various countries and
Oil body development in oilseeds their special attributes, 338
model, 13 CPO and PKO, 333
Oil crop breeding, 3–4 field experimental techniques
altered seed composition for health and data collection, 349–350
industrial applications, 19–20 mating designs, 348
milestones in, 10–11 randomized complete block design
oil content (RCBD), 349
botanical features of, 13–16 genetic conservation and study, 337
breeding for, 18–19 oil yield, breeding
genetics, 16–18 abiotic and biotic stress tolerance, 342
oil bodies and cytology of, 12–13 adaptability, 342
perspectives in harvestable/recoverable/realizable
biology, 21–22 yield, 341–342
technology, 20–21 potential yield, 341
utilization, 22 origin and domestication
Oil palm commercial dura pisifera hybrid
breeding achievements palm seed production, 335
cloning, 339 Deli Ds, 334
Dumpy.AVROS variety, 339–340 shell thickness, monogenic inheritance
tenera hybrid improvement, 338–339 of, 335
breeding, goals, 340 thin-shell tenera (T) palms, 334–335
desirable traits, priority list, 341 seed market, 360
oil quality, 343 companies and their estimated annual
breeding methods seed supply, 359
backcross breeding method, 345–346 seed processing
clonal propagation for, 346 depericarping and fresh seed quality
index and BLUP selection, 346–347 checking, 358
modified reciprocal recurrent heat treatment, 358
selection, 344–345 moisture adjustment and storage, 358
modified recurrent selection method resoaking and germination, 358
(MRS), 343–344 selection and dispatch, 358–359
recombinant inbred breeding, 346 somaclonal variation in, 350–351
542 Index
Oil palm (cont.) traditional line breeding, 106

varietal groups, 335 world consumption, 91
BPROs, derived and recombinant, 337 Olea europaea L., see Olive
Calabar, 337 Oleasters, see Wild olive
Deli and AVROS, 336 Oleic acid (OA), 343
Ekona, 336–337 de novo synthesis from glucose/carbon
Yangambi and La Me, 336 sources in humans, 37
world average yield, 3 Oleuropein in olive, 400
Oilseed rape Olive germplasm
breeding achievements cryopreservation, 415
erucic acid content, 98 haploid recovery and polyploids, 414
HOLLi phenotype, 100 protoplast culture, 414
lipid composition in, 99 somaclonal variation, 414
breeding goals Olive
disease resistance in, 103 breeding achievements, 407, 409
performance trials for identification clonal rootstocks, selection of, 408
of candidate varieties at breeding goals, 409
commercial breeding station, 102 breeding methods and techniques
seed and oil yield, potential and classical breeding, 409–410
stability, 101–103 clonal and sanitary selection, 410
seed components improvement, intervarietal crossing, 410–411
103–105 marker assisted breeding,
value and suitability, 104 411–412
gene pool breeding programs, 412
Brassica breeding material, 94 genetic manipulation, 413–414
genetic diversity in primary, 93–95 organogenesis and regeneration, 413
vernalization, 93 in vitro technologies, 414–415
generalized scheme for breeding of crop and production areas, importance
OP, 107 of, 397
genetic modification cultivation
fatty acid composition, 110 ancient and recent history of, 401
genetic variability and interspecific problems of, 397–399
hybridization, 95–98 genetic resources
genome analysis and marker-assisted natural diversity of, 405
selection subspecies, 407
biotic and abiotic stress, 114–116 taxonomy and distribution of, 404
genetic maps and QTL analysis, wild olive, 405–407
111–113 origin and domestication, 401–402
male sterility, 113 types
novel genomic tools, 116 oleuropein, 400
oil content and quality, 113–114 virgin olive oil, 399–400
synteny and arabidopsis, utilization varietal groups
of, 116–117 cultivars identification and
yellow seed character, 114 distribution, 403–404
hybrid breeding and cytoplasmic male cultivated olive germplasm,
sterility systems, 106, 108 402–403
male fertile and genic male sterile Orobanche resistance evaluation, 191
flowers, 109 Overdominance theory, 496–497
pathogens and pests, 103
seed production
hybrid, 117–118 P
requirements for, 117–119 Palm kernel oil (PKO), 333
tissue culture and haploid techniques, 109 Panama Tall clustering, 386–387
Index 543
Peanut breeding
breeding achievements, 294 Agrobacterium tumefaciens-mediated
cultivar Florunner, 295 genetic transformation, 462
disease resistance, 296–298 biological and genetic characteristics,
high oleic acid content, 295–296 457–459
breeding goals hybridization and pedigree method of
for farmers, 298–299 selection, 460–461
manufacturer and consumer, isoenzyme markers, 461
300–301 molecular cloning of, 462
seed producer/sheller, 299–300 morphological homogeneity, 455
breeding methods and techniques, and oil, culinary cultivars for,
301–302 463–464
gene sequencing in, 305–306 resistance against abiotic factors, 456
genetic resources resistance breeding, significance of,
ICRISAT collection, 294 455–456
marker development in, 303–304 seed color and oil content
molecular maps, 304–305 increasing, 457
origin and domestication, 289–292 yield, increase of, 455
reverse genetic technologies, 306 genetic resources and varietal groups,
seed production, 307 452, 455
transformation of, 306–307 culinary and industrial cultivars,
varietal groups, 292 separation of, 454
world production, 287 cultivars in E U, 453
Peredovik variety, 167 industrial utilization of, 450
See also Sunflower origin and domestication, 451–452
Phenotypic recurrent selection in sunflower Powdery mildew in sunflower, 164
breeders, 181 Prostaglandins eicosanoid family of
See also Sunflower metabolites synthesis from ARA
Phoma leaf spots by Leptosphaeria maculans and EPA, 37
in oilseed rape, 102 Prostate specific antigen (PSA), 474
resistance from B. rapa,96 Przemko cultivars, 463
in sunflower, 164 See also Poppy seeds
Phomopsis stem canker by Diaporthe PSR23 cuphea breeding line, 518
helianthi Pumpkin seed
in sunflower, 164, 191 breeding, goals of, 480
Physaria fendleri, 508 bush growth habit, 484–486
Phytophthora drechsleri,430 disease resistance, 486–487
Phytophthora root rot, 70 genetic base of oilseed pumpkins,
See also Soybean 487–488
Pink bollworms by Pectinophora increasing seed yield, 481
gossypiella okra-leaf trait seed size and number, 483–484
resistence, 267 yield, components, 481–482
Plus palms, 382–383 breeding programs, biotechnologies
Polish cultivar, 131 integration of, 487–488
See alsoBrassica napus spp. food crop, use, 469–470
Polyketide synthase (PKS) gene expression hull-less seed character, genetics, 475
in plant and yeast, 44 seed coat character and inheritance,
Poppy seeds, 449 studies, 476
alkaloid segregation of, 478–479
content of capsules, 456 single fruit, range of variation, 479
formation, studies, 461–462 Styrian oil pumpkin, crossing,
production, industrial cultivars for, 476–477
462–463 within-fruit variation of seed, 480
544 Index
Pumpkin seed (cont.) reduced and striped hull seeds, 432

hull-less seeded phenotype, origin of, breeding methods
470–471 backcrossing, 437
nutritionally relevant components hybridization, 438–440
carotenoids and chlorophylls, 473 pedigree breeding, 437
fatty acid content, distribution of, pure-line and mass selection, 437
471–472 recurrent selection programs, 438
oil, antioxidant activity of, 474–475 breeding programs
sterol and secoisolariciresinol (SECO) Agrobacterium tumefaciens-mediated
content in, 472 transformation and, 440
symptomatic bph, treatment with molecular farming, 442
b-sitosterol, 475 oil-bearing tissue, 441
Pusa Selection 1-3, 463 oil-body membrane, recombinant
See also Poppy seeds proteins produced in, 441
Pustovoit’s method of reserves, 184 SemBioSys genetically transforms,
440–441
crossing techniques, 433
R crossed seeds, head of, 436
Rajhans Chetak cultivars, 463 developmental stage for, 434
See also Poppy seeds elongated styles, 434–435
Random-mated population in sunflower emasculation by sliding corolla tube
breeders, 182 upwards, 434
See also Sunflower mass-emasculation technique,
Raphanus sativus beet cyst nematodes on, 98 436–437
Reciprocal full-sib selection method in pollination, 434–436
sunflower breeders, 182–183 genetic resources
Recurrent selection (RS) germplasm collections, 428
maize breeding, 499 germplasm management unit
Reniform nematodes by Rotylenchulus (GMU), 426
reniformis, cotton diseases, 268 International Board for Plant
Reverse high-oil (RHO) and reverse low-oil Genetic Resources (IBPGR)
(RLO), 501 guidelines, 427
Rhizopus head rot by Rhizopus spp. in origin and domestication, 423–424
sunflower, 164 seed production, 442–443
Ricinus communis L., see Castor seed species groupings breeding,
Roguing in sunflower hybrid seed 424–426
production, 214 Sclerotinia blight by Sclerotinia minor Jagger
Root-knot nematodes by Meloidogyne in peanuts, 288
incognita, cotton lines, 268 Sclerotinia stem rot by Sclerotinia
Russia sclerotiorum
scientific sunflower breeding, 167 in oilseed rape, 101, 135
sunflower cultivation as oil crop, 167 resistance by oilseed rape, 115
Rust by Puccinia arachidis Speg. in in sunflower, 164
peanuts, 288 SemBioSys genetically transforms safflower
tissue, 440–441
Sequence-characterized amplified region
S (SCAR) markers in B. rapa,141
SAD enzyme in sunflower, 200 Sinapis arvensis,98
Safflower seed Single-cross hybrids in sunflower, 186
breeding achievements, 429–430 b-Sitosterol, 475
breeding goals, 430, 433 in soybean oil, 84
partial hull seeds and white seeds Soma cultivars, 463
of, 431 See also Poppy seeds
Index 545
Soybean Mexican lines of, 65

accessions new biotechnologies into breeding
geographical distribution, 63 programs, integration, 80
breeding accomplishments, 66 germplasm and, 81–83
conventional breeding strategies, 67 sterols and tocopherol, 84
diseases and pests, resistance for, 70 origin, 58–59
ethyl methanesulfonate (EMS) plant phenlogy and morphology changes
mutagenized with, 68–69 by domestication, 60
fan 1,fan 2 and fan 3, 68 seed
GMO soybeans for food production, 9 desaturase (SAD) genes and
69–70 stearic acid, 34
high oleic (HO) soybean, 70 desaturase and thioesterase
linolenic acid content research, 67–68 activities, 33
marker-assisted-selection, 71 FAD2 expression modulation, 34
Roundup Ready1(RR) soybean, 69 Fusarium moniliforme and ALA
Roundup Ready2yield1, 69 concentrations, 39
world oil production, 68 improvements in, 58
breeding methods and techniques modification by raising oleic acid
advancing toward homozygosity, content, 32
77–78 palmitate content rehulated by FatB
gain from selection, 74–75 genes, 33
intra-cultivar variation, 79 polyunsaturated fatty acids source, 37
new technology in, 79–80 seed-specific silencing of FATB
parent and population structure, genes, 34
75–77 taxonomic classification of, 58
participatory plant breeding, 78 varietal groups of
selection among breeding lines, 78–79 BSR 101, cross between, 62
sources of, 75 genetic distance within progenitors/
brown stem rot caused by Phialophora cultivars, 61–62
gregata, 64 genetic diversity, evaluation of, 61, 64
chromosome size and study, 61 genetic male sterility and heterosis
constituents of, 58 expression in, 62
consumer acceptance and HybSoy 1 and HybSoy 2, 62
consumption, 57 QTL environment interaction, 62
C18:3 soybean development, 83 world production of, 57
cultivated, ancestors of, 59 Spring-sown oilseed rape, 93
current goals of breeding S1 progeny/testcross evaluation in sunflower
fatty acid modification, 71–72 breeders, 182
increased polyunsaturates, 73 Sri Lanka coconut breeding program, 388
increasing nutraceuticals in seed, Sri Lanka Tall, 384
73–74 See also Coconut palm
monounsaturated fatty acid State Agricultural Experiment Stations
content, 72 (SAES), 67
reduction and increase in saturates, 72 Stearic acid vegetable oil modification and
seed oil concentration, 71 stearoyl-ACP thioesterase, 34
trans-fat reduction, 72–73 Stearidonic acid (STA), 39
cyst nematode caused by Heterodera in commercial oil seed crops, 40
glycines, 64 in soybean, 40
gene pool in, 60 Stem canker by Leptosphaeria maculans, 101
before and after genetic See also Oilseed rape
bottlenecks, 65 Stigmasterol in soybean oil, 84
Glycine spp. study, 60 Strubes Blauer cultivars, 462
industrial use of, 47 See also Poppy seeds
546 Index
Styrian oil pumpkin, 476–477 embryogenesis, 205

sclerenchyma layer in hull-less Type 2 and fatty acid profile, 200
Type 3seeds, 478 herbicides resistance, 206
Sunflower oil content, 199
breeding achievements self-incompatibility and seed
high oil germplasm in USSR, dormancy, 205
development of, 167 tocopherols, 202–204
inbred-hybrid method, 167–168 morpho-physiological traits after
new oil types development, 168–169 breeding
breeding methods abiotic stresses and, 178–179
existing genetic variation use, 180 broomrape resistance, 177–178
hybrid cultivars improvement, 184–186 disease resistance, 176–177
hybridseed production, 186–187 fatty acid and tocopherol profiles
mutagenesis, 181 in, 173
open pollinated cultivars flowering and maturity dates, 170–171
improvements, 183–184 head size, shape and inclination, 170
source populations improvement, herbicide resistance, 179
181–183 insect resistance, 178
consumer acceptance, 155 male sterility, 171
field plot techniques for cultivar nonoil seed seeds, 179
evaluation, 188 oil, protein and fiber contents,
functional markers, 198 171–172
gene-targeted markers and maps oil quality, 172
based on oleic acid content, 175
ESTs, 197 plant height, 169–170
sequenced RFLP-cDNA probes, 197 pollen self-compatibility and flower
genetic markers and linkage maps in, 192 characteristics, 171
germplasm characterization, 198–199 resistance to bird depredation, 178
germplasm collection and maintenance seed meal quality, 175–176
core collections, 161 origin and domestication, 156
ex situ world collections, 160 hybrid varieties, 159
genetic stock collections, 161 infrageneric classification, 157–158
in situ resources, preservation of, oleic acid content, 159
160–161 ornamental cultivars, 159
germplasm evaluation random DNA markers and maps based
agronomic and physiological traits, 162 on
cytoplasmic male sterility (CMS), AFLP and RAPD markers, 195
162–163 DALP markers, 196
disease and insect resistance, 164–165 RFLP markers, 192
oil and protein, content and SCAR markers, 195
quality, 166 SSRs/microsatellites, 196
greenhouses and off-season nurseries, 189 seed production, 211
human activities impact on cultivation commercial hybrid seed production,
of, 161 213–214
interspecific hybridization, 187–188 maintenance and increase of parental
linkage genetic maps for, 193–194 lines, 212
marker assisted selection, 206–210 seed quality evaluation, 189–190
molecular mapping of inherited and seed yield after breeding, 169
complex traits selfing and artificial hybridization, 187
abiotic stresses, 205 techniques for disease resistance and
days to flowering, 205 broomrape evaluation, 190–191
developmental and agronomic transgenic breeding, 210–211
traits, 204 Switchback high-oil (SHO), 501
Index 547
T soybean
Tagnanan Tall, 384 breeding programs, 64
See also Coconut palm early breeding, 67
Talent hybrid cultivar of oilseed rape, 108 genetic diversity in, 64
Target region amplification polymorphisms Plant Variety Protection (PVP)
(TRAPs) for sunflower, 197 Act, 67
TC Blend1 seed corn method, 503 USDA-ARS in cultivar
Tebona cultivars, 463 development,67
See also Poppy seeds variability in, 64
Test cross/half-sib progeny recurrent sunflower germplasm
selection in sunflower breeders, 182 U.S. National Plant Germplasm
Tetracosahexaenoic acid (THA) and System (NPGS), 160
tetracosapentaenoic acid (TPA), 43 USDA/ARS
Tetraploid plants, 414 flax improvement research, 240
Tevere cultivars, 408 National Arid Land Plant Genetic
See also Olive Resources Unit, 509
Thromboxanes eicosanoid family of
metabolites synthesis from ARA
and EPA, 37 V
Tifguard cultivar, 298 Vavilov All-Union Scientific Research
See also Peanut Institute (VIR) and sunflower
Tobacco plant, GLA and STA synthesis, 40 germplasm, 160
Tobacco streak virus disease in Vegetable oils
sunflower, 177 consumption, 1–2
Tocopherols modification for non-food purposes
in soy-based foods, 74 high oleic acid soybean oil, 45–46
in soybean oil, 84 metabolic engineering of soybean
in sunflower oil, 173 for production of oils with
g-and tocopherol in pumpkin, 472 high-value industrial fatty acids,
Tomato spotted wilt virus (TSWV) in 46–49
peanuts, 288 non-food uses of plant oils, 44–45
TopCross1method, 503 vernolic acid in, 47
Tosca winter oilseed rape varieties, 96 Verticillium wilt
Two-step embryo culture procedure in resistence by RS B. napus genotypes, 96
sunflower, 188 in sunflower, 164
Virgin olive oil, 399–400
U
United Soybean Board (USB), Better Bean W
Initiative (BBI) program, 71–72 Whitefly by Bemisia tabaci, cotton host plant
United States resistance, 267
cottonseed White mold/stem rot by Sclerotium rolfsii
belt, 267 Sacc. in peanuts, 288
collection of, 266 White rust by Albugo candida,135
upland cotton acreage planted to Wild olive
genetically engineered (GE) evolution of, 405–406
varieties planted by state and interpopulation, levels of, 406–407
across, 274 for olive oil extraction, 401–402
peanut studies, 406
market types of, 292–293 World acreage and average annual seed/fruit
production in, 288 yield of major oil crops, 2
safflower seed, hybridization of, WRKY transcription factors for defense
438–439 against sclerotinia rot, 115
548 Index
Y Z
Yellow- seeded rapeseed materials by Zeno Wintermohn cultivars, 464
interspecific crosses between See also Poppy seeds
B. rapa and B. oleracea, 97 Zero-erucic acid germplasm in B. juncea, 131

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Oil Crops

HANDBOOK OF PLANT BREEDING

ISBN 978-0-387-77593-7 e-ISBN 978-0-387-77594-4

Library of Congress Control Number: 2009930437

# Springer ScienceþBusiness Media, LLC 2009

Printed on acid-free paper

Springer is part of Springer Science+Business Media (www.springer.com)

When one is privileged to participate long enough in a professional capacity,

for food use. The most World Vegetable Oil Consumption

World Industrial Use Vegetable

renewed interest in Food Use 20

figure, the market forces USDA FAS, Jan 2009

vegetable oils appear to have established a temporary equilibrium at about 80%

depends upon a renaissance in quantitative genetics and the application of those

Raleigh, North Carolina Richard F. Wilson

Vienna, Austria Johann Vollmann

1 Oil Crop Breeding and Genetics . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1

2 Modifying Vegetable Oils for Food and Non-food Purposes . . . . . . . 31

5 Other Brassicas . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 127

11 Oil Palm. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 333

16 Hull-Less Oil Seed Pumpkin . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 469

17 Maize for Oil . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 493

18 New Crops Breeding: Lesquerella. . . . . . . . . . . . . . . . . . . . . . . . . . . . 507

Howard G. Damude DuPont Experimental Station, Crop Genetics Research

Hans-Henning Mündel West View Mall, Lethbridge, AB T1K 6X4, Canada,

Leonardo Velasco Institute for Sustainable Agriculture (CSIC), Alameda del

Johann Vollmann and Istvan Rajcan

Oil crops have considerably gained in importance to world agriculture and

J. Vollmann, I. Rajcan (eds.), Oil Crops, Handbook of Plant Breeding 4, 1

(FAOSTAT 2007). Subsequently, the projected rise in vegetable oil consump-

dual-purpose linseed cultivars have occasionally been suggested (Rennebaum

1.2 Domestication and Genetic Diversity

1.2.1 Domestication of Oil Crops

The domestication status of oil crops is fairly divergent depending on their

1.2.2 Oil Crop Germplasm

The availability of germplasm with sufficient genetic diversity is essential for a

System holds significant collections of soybean, peanut and cotton accessions in

1.2.3 Genetic Diversity in Oil Crops – Selected Examples

Korea, respectively. In a diversity study based on AFLP markers, Ude et al.

pools of accessions with sufficiently large genetic distance need to be

1.3 Recent Milestones in Oil Crop Breeding

later transferred to Brassica napus in France; cms in Chinese

1.4 Specific Breeding Objectives

1.4.1 Oil Content

very large oil bodies (5–50 mm in diameter) are found in non-desiccating

1.4.1.2 Botanical Features of Oil Content

Fig. 1.3 Low (left) and high

1.4.1.3 Genetics of Oil Content

1.4.1.4 Breeding for Oil Content

been presented. Thus, high-throughput technologies for screening large

1.4.2 Altered Seed Composition for Health and Industrial

1.5 Perspectives in Oil Crop Breeding

genetic diversity, prediction of hybrid performance, QTL analysis, marker

On the level of biology, issues such as maintenance of genetic diversity, the

increasing diversity while being challenged by the need to maintain an accep-

Edgar B. Cahoon, Thomas E. Clemente, Howard G. Damude,

E.B. Cahoon (*)

J. Vollmann, I. Rajcan (eds.), Oil Crops, Handbook of Plant Breeding 4, 31

concomitantly reduces the polyunsaturated fatty acids. Partial hydrogenation

2.2.2 Conventional Approaches to Fatty Acid Modification

In soybean seed the content of palmitate is regulated by a palmitoyl thioes-

Implementing the tools of biotechnology novel oil traits can be achieved in a

Targeted perturbation of FAD2 alleles in a seed-specific fashion in